Platelet Protection Solution Having Beta-Galactosidase and Sialidase Inhibitors

ABSTRACT

The present invention relates to a platelet protection solution (PPS) having an amount of one or more β-galactosidase inhibitors with or without an amount of one or more sialidase inhibitors, and optionally one or more glycan-modifying agents; and one or more of PPS components that include a salt, a citrate source, a carbon source, or any combination thereof.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/110,640, entitled, “Platelet Protection Solution Having aBeta-Galactosidase Inhibitor” by Qiyong Peter Liu et al., filed Feb. 2,2015; and is a Continuation-In-Part of U.S. application Ser. No.14/047,689, filed Oct. 7, 2013, entitled “Platelet Additive SolutionHaving A Beta-Galactosidase Inhibitor;” which claims the benefit of U.S.Provisional Application No. 61/813,885, filed Apr. 19, 2013, entitled,“Platelet Additive Solution Having a Platelet Enhancing Agent;” and U.S.Provisional Application No. 61/710,273, filed Oct. 5, 2012, entitled,“Platelet Additive Solution Having a Sialidase Inhibitor and/or aBeta-Galactosidase Inhibitor;” and application Ser. No. 14/047,689 is aContinuation-In-Part of U.S. Application filed Ser. No. 14/856,179,filed Sep. 16, 2015, which is a continuation of U.S. application Ser.No. 13/474,627, entitled “Platelet Storage and Reduced BacterialProliferation In Platelet Products Using A Sialidase Inhibitor” by Liuet al., filed May 17, 2012, which is a continuation of U.S. applicationSer. No. 13/474,473, entitled “Increased In Vivo Circulation Time ofPlatelets After Storage With A Sialidase Inhibitor” by Liu et al., filedMay 17, 2012, and claims the benefit of U.S. Provisional Application No.61/613,876, filed Mar. 21, 2012; U.S. Provisional Application No.61/613,837, filed Mar. 21, 2012; U.S. Provisional Application No.61/503,984, filed Jul. 1, 2011; and U.S. Provisional Application No.61/487,077, filed May 17, 2011.

The entire teachings of the above applications are incorporated hereinby reference.

GOVERNMENT SUPPORT

The invention was made with Government support under grant No. HL089224awarded by the National Institutes of Health. The Government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Collected platelets intended for transfusion are highly perishable.Platelets are non-nucleated bone marrow-derived blood cells that protectinjured mammals from blood loss by adhering to sites of vascular injuryand by promoting the formation of plasma fibrin clots. Humans depletedof circulating platelets by bone marrow failure suffer from lifethreatening spontaneous bleeding, and less severe deficiencies ofplatelets contribute to bleeding complications following trauma orsurgery.

As the count of circulating platelets falls (e.g., ˜70,000 μL), perpatients become increasingly susceptible to cutaneous bleeding. Patientswith platelet counts of less than 20,000 per μL are highly susceptibleto spontaneous hemorrhage from mucosal surfaces, especially when thethrombocytopenia is caused by a bone marrow disorder or failure.Platelet deficiencies associated with bone marrow disorders such asaplastic anemia, acute and chronic leukemia, metastatic cancer, anddeficiencies resulting from cancer treatment such as ionizing radiationor chemotherapy all contribute to a major public health problem.Patients that suffer from thrombocytopenia associated with majorsurgery, injury and sepsis also require significant numbers of platelettransfusions.

A major advance in medical care half a century ago was the developmentof platelet transfusions to correct such platelet deficiencies,resulting in about 2.6 million platelet transfusions in the UnitedStates per year at current transfusion rates. However, plateletscollected for transfusion are highly perishable because, upon storage ator below room temperature, they quickly lose in vivo hemostaticactivity. Hemostatic activity broadly refers to the ability of apopulation of platelets to mediate bleeding cessation.

Platelets, unlike all other transplantable tissues, do not toleraterefrigeration and disappear rapidly from the circulation of recipientsif subjected to even very short periods of chilling. Importantly, thecooling effect that shortens in vivo platelet survival is thought to beirreversible and, therefore, cooled platelets become unsuitable fortransfusion. One of the first visible effects of platelet impairment isan irreversible conversion from a discoid morphology towards a sphericalshape, and the appearance of spiny projections on the surface ofplatelets due to calcium dependent gelsolin activation andphosphoinositide-mediated actin polymerization. When platelets areexposed to temperatures lower than 20° C., they rapidly undergo suchshape modifications.

The need to keep platelets at room temperature prior to transfusion hasimposed a unique set of costly and complex logistical requirements onplatelet storage. Because platelets are metabolically active at roomtemperature, they require constant agitation in gas permeable containersto allow for the exchange of gases to prevent the toxic consequences ofmetabolic acidosis. Room temperature storage conditions result inmacromolecular degradation and reduced hemostatic functioning ofplatelets, a set of defects known as the “platelet storage lesion”(PSL). In addition, storage at room temperature encourages the growth ofbacteria, thereby creating a higher risk of bacterial infection, whicheffectively limits the duration of such storage to about 5 days. Thesebacteria include endogenous bacteria as well as skin-derived onesassociated with venipuncture. In this regard, bacterial contamination ofplatelets is by far the most frequent infectious complication of bloodcomponent use. At current rates, from one in 1,000 to one in 2,000 unitsof platelets are contaminated with bacteria at a level sufficient topose a significant risk to the recipient.

Thus, there remains a pressing need to develop agents, solutions andmethods to (i) improve or prolong in vivo hemostatic activity of humanplatelets upon storage at or below room temperature, (ii) stabilizeplatelets during storage to prevent their premature clearance fromcirculation following transfusion, and/or (iii) more significantly,inhibit bacterial proliferation during room temperature plateletstorage.

SUMMARY OF THE INVENTION

The present invention relates to a platelet protection solution (PPS)that includes an amount of one or more β-galactosidase inhibitors withor without an amount of one or more sialidase inhibitors and,optionally, one or more glycan-modifying agents; and one or more PPScomponents that include a salt (e.g., sodium source, a chloride source,a potassium source, a magnesium source, a calcium source, or acombination thereof), a citrate source (e.g., monosodium citrate,disodium citrate, trisodium citrate, citric acid, or a combinationthereof), and/or a carbon source (e.g., acetate, glucose, sucrose, orany combination thereof). For example, the PPS can include an amount ofone or more of any of the following: β-galactosidase inhibitors;β-galactosidase inhibitors and sialidase inhibitors; β-galactosidaseinhibitors and glycan-modifying agents; or β-galactosidase inhibitors,sialidase inhibitors and glycan-modifying agents. The PPS, in anembodiment of the present invention, is maintained at a pH rangingbetween about 6.4 and about 7.6. In one embodiment, the PPS of thepresent invention further includes a phosphate source (e.g., sodiummonophosphate, diphosphate, triphosphate or a combination thereof). Anacetate source can include, for example, sodium acetate, potassiumacetate, magnesium acetate or a combination thereof. In an aspect, thesodium source can be sodium chloride, sodium citrate, sodium acetate,sodium phosphate or a combination thereof. Similarly, the chloridesource can be sodium chloride, magnesium chloride, potassium chloride ora combination thereof. The potassium source, in an example, can bepotassium chloride, potassium citrate, potassium acetate, potassiumphosphate, potassium sulfate or a combination thereof. Examples ofsources of magnesium include magnesium chloride, magnesium citrate,magnesium sulfate and a combination thereof. In an embodiment, thecalcium source encompasses calcium chloride, calcium acetate, calciumcitrate or a combination thereof.

In a particular embodiment, the PPS of the present invention includes anamount of one or more β-galactosidase inhibitors (e.g., between about0.001 mM to about 10 mM) with or without an amount of one or moresialidase inhibitors (e.g., between about 0.001 mM to about 10 mM) and,optionally, one or more glycan-modifying agents; a sodium source in anamount between about 100 mM and about 300 mM; a chloride source in anamount between about 40 mM and about 110 mM; a citrate source in anamount between about 2 mM and about 20 mM; an acetate source in anamount between about 10 mM and about 50 mM; a phosphate source in anamount between about 5 mM and about 50 mM; a potassium source in anamount between about 0.5 mM and about 10 mM; a magnesium source in anamount between about 0.5 mM and about 5.0 mM; a calcium source in anamount between about 0 mM (e.g., 0.1 mM) and about 2.5 mM and a glucosesource in an amount between about 0 mM (e.g., 0.1 mM) and about 30 mM.An alternative to the embodiment above has the same components and ismaintained at a pH of between about 6.4 and about 7.6 (e.g., about 7.1to about 7.4, or about 7.2).

In yet another embodiment, the present invention pertains to plateletcompositions having isolated platelets; the PPS of the presentinvention; and plasma, wherein the platelet composition is maintained ata pH ranging between about 6.4 and about 7.6. In an aspect, the plasmais present in an amount between about 1% and about 50% by volume (e.g.,between 20% and 40% plasma, or about 30% plasma). In yet anotherembodiment, the platelet protection solution is present in an amountbetween about 50% and about 99% by volume.

The present invention further relates to a bag or container suitable forplatelet storage having the PPS of the present invention. The bag orcontainer can further include isolated platelets that can be maintainedat a pH ranging between about 6.4 and about 7.6.

The present invention relates to a method of storing platelets, whereinisolated platelets are obtained from one or more donors. The methodincludes the steps of contacting the isolated platelets with the PPSdescribed herein. The β-galactosidase inhibitor can be, e.g.,1-deoxygalactonojirimycin (DGJ); 1-deoxygalactonojirimycin HCl,N-(n-butyl)deoxygalactonojirimycin; N-(n-nonyl)deoxygalactonojirimycin;5-deoxy-L-arabinose; galactostatin bisulfite;3′,4′,7-trihydroxyisoflavone; D-ribonolactone;N-octyl-4-epi-β-valienamine; phenylethyl β-D-thiogalactopyranoside;difluorotetrahydropyridothiazinone; 4-aminobenzyl1-thio-β-D-galactopryranoside; a combination thereof; or apharmaceutically acceptable salt thereof. The sialidase inhibitor can bee.g., fetuin; 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA);Oseltamivir(ethyl(3R,4R,5S)-5-amino-4-acetamido-3-(pentan-3-yloxy)-cyclohex-1-ene-1-carboxylate);Zanamivir((2R,3R,4S)-4-guanidino-3-(prop-1-en-2-ylamino)-2-((1R,2R)-1,2,3-trihydroxypropyl)-3,4-dihydro-2H-pyran-6-carboxylicacid); Laninamivir((4S,5R,6R)-5-acetamido-4-carbamimidamido-6-[(1R,2R)-3-hydroxy-2-methoxypropyl]-5,6-dihydro-4H-pyran-2-carboxylicacid); Peramivir((1S,2S,3S,4R)-3-[(1S)-1-acetamido-2-ethyl-butyl]-4-(diaminomethylideneamino)-2-hydroxy-cyclopentane-1-carboxylicacid); any combination thereof or a pharmaceutically acceptable saltthereof. In an embodiment, the sialidase inhibitor is the sodium salt of2,3-dehydro-2-deoxy-N-acetylneuraminic acid.

The method allows isolated platelets to be stored for a period of about1 to about 21 days. The isolated platelets are stored a temperature ofbetween about 1° C. and about 25° C. (e.g., about 2° C. to about 24°C.). The method, in an embodiment, includes the steps of cooling theplatelet composition to a temperature below room temperature; storingthe platelet composition for a period of time; and then rewarming theplatelet composition back to room temperature. In an aspect, thepopulation of platelets is treated with the β-galactosidase inhibitor,or both with the β-galactosidase inhibitor and the sialidase inhibitorwithin a time period, wherein the time period is in a range betweenabout 1 minute to about 48 hours.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-C are schematics depicting a sialylated platelet containingintracellular sialidase and sialidase-containing bacteria. (FIG. 1A)Both bacterial and platelet derived sialidases remove sialic acid fromplatelet surfaces, leading to the formation of platelets with impairedfunction (1). The released sialic acids support the proliferation ofcontaminating bacteria (short-dashed line and 2), which leads toplatelet activation (3), formation of platelet-bacteria aggregates (3),and biofilm formation (long-dashed line and 4). (FIG. 1B) Desialylatedplatelets are recognized and removed from the circulation by phagocytesupon transfusion. (FIG. 1C) Addition of the sialidase inhibitor DANA(sodium salt of 2,3-dehydro-2-deoxy-N-acetylneuraminic acid) inhibitsthe sialidase activities derived from platelets and bacteria, andprevents platelet desialylation so that platelets are not recognized byphagocytic cells after transfusion.

FIG. 2 is a bar graph showing that human platelets lose sialic acidduring storage at 4° C. Platelet concentrates (A: Donor A and B: DonorB) were stored at 4° C. for 5 days in the absence of exogenousnucleotide sugar (a), in the presence of CMP-sialic acid (CMP-SA) andUDP-galactose (UDP-gal) (b) or UDP-gal alone (c). Sialic acid content ofplatelets at day 0 was set to 100%.

FIGS. 3 (A) (B) & (C) are line graphs showing that the human plateletsialidase surface activity increases following cold storage. (A) depictsthe analysis of fresh platelets, with or without permeabilization. (B)depicts the analysis of fresh intact platelets (Donors A and B) at pH 5and 6. (C) depicts the corresponding analysis of intact platelets(Donors A and B) after storage at 4° C. for 5 days.

FIG. 4 shows immunofluoresence micrographs of fixed, non-permeabilized,resting room temperature (RT) (left panels) and refrigerated (rightpanels) human platelets demonstrating the presence of sialidase Neu3,but not Neu1, on their surfaces. Refrigeration (48 h) of plateletsincreases sialidase (Neu1) surface fluorescence, i.e, exposure.Anti-Neu1 antibody was used in the upper panels. Anti-Neu3 antibody wasused in the lower panels.

FIG. 5 is a graph showing that mouse platelet sialidase surface activityincreases following 48 h cold storage and rewarming. Platelet-derivedsialidase activity was measured in fluorescence (Absorption Intensity(AI)) over 0-2.5 h at room temperature. Platelet storage at coldtemperatures (4° C., darker circles) was compared with fresh platelets(RT, lighter circles). As a control, sialidase activity (Clostridiumperfringens (Component H)) was measured over the same time period(inset).

FIG. 6 is a bar graph showing that fetuin competes for sialidase surfaceactivity during platelet storage and thus inhibits the hydrolysis ofsialic acid from platelet glycans. The left pair of bars represents theβ-galactose exposure on fresh platelets (0) in the absence (Control) orpresence of fetuin (Fetuin). The right pair of bars represents theβ-galactose in the absence (Control) or presence of fetuin followingplatelet refrigeration for 48 h. Sialic acid loss, i.e., β-galactoseexposure, is measured by RCA I binding.

FIG. 7 is a graph showing that the sialidase inhibitor DANA increasesmouse platelet life span in vivo. The bottom line represents the controlplatelet life span (Control). The top line represents the platelet lifespan upon addition of DANA (DANA).

FIG. 8 is a schematic that shows (A) the structure of the primary GPIbαstructure and O- and N-linked glycans. (B) shows the structure andbiosynthetic modifications of terminal Galβ1, 4GlcNAc(lactosaminoglycan/LacNAc) and the Core-1 O-glycan.

FIG. 9 shows that human platelets contain the sialidases Neu1 and Neu3by Western blot analysis of total platelet lysates.

FIG. 10 shows that human platelets release Neu1 into plasma uponlong-term refrigeration as analyzed by Western blot. Platelets and theircorresponding plasma were analyzed at day 0 and following plateletrefrigeration for 1, 2, and 5 days.

FIG. 11 (A) depicts the characterization of plateletglycosyltransferases (GTs).

Human total platelet lysates were subjected to SDS-PAGE and wereimmunoblotted with monoclonal antibodies: anti-GalNAc transferases(GalNAc-T1, -T2, -T3), β4Gal-Transferase1 (β4Gal-T1), andsialyltransferase ST3Gal-1 (B) Platelets secrete GTs. Resting plateletswere maintained at 37° C. or activated via the thrombin receptor PAR-1with 25 μM TRAP, for 5 min. Maximal release was observed after 1 min.The Enzymatic Activity in counts per minute (CPM) was measured in thepelleted platelet fraction (P), or in their corresponding bathing media(M). The media was clarified at 100,000×g for 90 min to eliminatemicroparticles prior to activity measurements.

FIG. 12 depicts that endogenous platelet sialyltransferases incorporatesialic acid into platelet surface receptors. (A) Active human platelets'surface sialyltransferase incorporated FITC-conjugated CMP-SA (FITC-SA)into resting (dotted line) or TRAP-activated platelets. FITC alone(Control) was added to resting (dotted line) or TRAP activated platelets(solid line). (B) shows immunoblots of lysates from resting (Rest) orTRAP-activated platelets (TRAP) treated with FITC (F), FITC-CMP-sialicacid (S), or left untreated (−) and detected with antibodies to FITC,GPIbα, αIIb, and vWf. The blots shown are representative of twoexperiments. Actin is shown as a loading control.

FIG. 13 shows that platelets lose GPIbα and GPV receptors during storageat room temperature (A) or under refrigeration (B). Expression of mousevWf receptor complex components (GPIbα, GPIbβ, GPIX, GPV), GPVI andα_(IIb)β₃ was measured by flow cytometry before and after plateletstorage in the cold at the indicated time points. Results are expressedas means±SD, n=5. Glycoprotein expression on freshly isolated plateletswas set as 100%.

FIG. 14 shows that inhibition of metalloprotease-mediated GPIbα sheddingalone does not improve mouse platelet recovery and survival. (A) GPIbαand (B) GPV surface expression were assessed by flow cytometry.Wild-type mouse platelet rich plasma was stored for 0, 24, and 48 h at4° C. in the presence of DMSO (Control) or 100 μM of themetalloproteinase inhibitor GM6001 (n=6). Surface expression of (C)GPIbα and (D) GPV was determined by flow cytometry on freshly isolatedor 24 and 48 h refrigerated platelet rich plasma from TACE^(+/+) andTACE^(ΔZn/ΔZn) mice. Results are the mean±s.e.m. n=5. (C, Inset)Immunoblot for GPIbα in lysates from TACE^(+/+) and TACE^(ΔZn/ΔZn)platelets stored for 3, 24, and 48 h in the cold. (E)Fluorescently-labeled (5-chloromethyl fluorescein diacetate, CMFDA)fresh PRP (RT) or platelets from stored platelet rich plasma in theabsence (48 h) or presence of 100 μM GM6001 (48 h+GM6001), were infusedinto wild-type mice (10⁸ platelets/10 gm of body weight). Blood wasdrawn at the indicated time points, and platelets were immediatelyanalyzed by flow cytometry. Results are mean percentage CMFDA-labeledplatelets±s.e.m. The percentage of CMFDA positive fresh platelets attime 5 min post-transfusion was set as 100%. n=5. *P<0.05. Cold-storedplatelets are compared. (F) Fluorescently-labeled (CMFDA) freshplatelets (TACE^(+/+) RT and TACE^(−/−) RT) or platelets from storedplatelet rich plasma (TACE^(+/+)48 h and TACE^(−/−)48 h) were infusedintravenously into wild type mice (10⁸ platelets/10 gm of body weight).Blood was drawn at the indicated time points, and platelets wereimmediately analyzed by flow cytometry. Results are mean percentageCMFDA-labeled platelets±s.e.m. The percentage of CMFDA positive freshTACE^(+/+) platelets at 5 min post-transfusion was set as 100%. n=5.

FIG. 15 shows that sialidase-treated TACE^(ΔZn/ΔZn) platelets arerapidly cleared from the circulation. (A) Flow cytometric analysis ofβ-galactose exposure on glycoproteins, as detected with ECL FITC-labeledlectin is shown. Lectin binding to TACE^(+/+) (white bars) orTACE^(ΔZn/ΔZn) (hatched bars) platelets treated or not treated withα2-3,6,8,9-sialidase (Neu). The ratio of mean fluorescence intensitybinding to untreated TACE^(+/+) platelets is shown. Histograms reportthe mean±s.e.m. for three separate experiments. *P<0.05, **P<0.01,***P<0.001. (B) GPIbα, GPV, and α_(IIb)β₃ surface expression wasassessed by flow cytometry. TACE^(+/+) (not shown) and TACE^(ΔZn/ΔZn)platelets were treated with sialidase (5 mU/mL) (hatched bars) or not(white bars). Results are expressed relative to the amount of GPIbα onTACE^(ΔZn/ΔZn) platelets (mean % relative to control±s.e.m.). n=3. (C)Fresh, room temperature and fluorescently-labeled (CMFDA) TACE^(+/+) andTACE^(ΔZn/ΔZn) platelets treated with α2-3,6,8,9-Sialidase (5 mU/mL)(filled symbols) or left untreated (open symbols) were infusedintravenously into TACE^(+/+) mice (10⁸ platelets/10 g of body weight).Blood was drawn at the indicated time points, and the platelets wereimmediately analyzed by flow cytometry. Results are expressed as themean percentage CMFDA-labeled platelets±s.e.m. The percentage of CMFDApositive untreated TACE^(+/+) platelets at 5 min post-transfusion wasset as 100%. Each point represents 4 mice. n. s. not significant,***P<0.0001. Sialidase treated TACE^(+/+) and TACE^(ΔZn/ΔZn) werecompared.

FIG. 16 is a bar graph showing that neuraminidase treatment of plateletsincreases β-galactose exposure (loss of sialic acid) as measured by ECLfluorescence lectin binding. Data is from flow cytometric analysis ofβ-galactose or β-GlcNAc exposure on platelet glycoproteins, as detectedwith ECL I (open bars) or s-WGA (closed bars) FITC-labeled lectins.Results have been obtained from lectin binding to fresh mouse plateletsin the presence and absence of α2-3,6,8,9-Sialidase from A. ureafaciens(Neu) at the indicated concentrations, n=5.

FIG. 17 is a bar graph showing the dose dependent loss of platelet GPIbαand GPV receptors with increasing neuraminidase concentrations. GPIbαand GPV surface expression on freshly isolated mouse platelets wasassessed by flow cytometry. Surface receptor expression in the presenceand absence of α2-3,6,8,9-sialidase (Neu) at the indicatedconcentrations is shown. The mean fluorescence of receptor expression attime 0 was set as 100%. n=4.

FIG. 18 is a bar graph showing that DANA inhibits the exposure ofβ-galactose by neuraminidase treatment. Data is from flow cytometricanalysis of β-galactose or β-GlcNAc exposure on mouse plateletglycoproteins, as detected above in the presence (Neu) and absence(Control) of 5 mU α2-3,6,8,9-sialidase (Neu) and the competitivesialidase inhibitor DANA (Neu+DANA). n=4.

FIG. 19 is a bar graph showing that DANA inhibits the loss of plateletGPIbα, GPV, GPIX, and α_(IIb)β₃ receptors induced by neuraminidasetreatment. Surface receptor expression (GPIbα, GPV, GPIX, and α_(IIb)β₃)was measured by flow cytometry on mouse platelets in the presence (barshatched with negatively sloping lines) and absence (open bars) of 5 mUα2-3,6,8,9-sialidase. Receptor expression on platelets treated withsialidase and DANA is also shown (bars hatched with positively slopinglines). The mean fluorescence of receptor expression on untreatedplatelets was set as 100%. n=4.

FIG. 20 depicts a non-reduced immunoblot of total platelet lysates(INPUT), supernatants (SUPERNATANT) and the corresponding platelets'pellet (PELLET) showing that DANA inhibits the loss of platelet GPIbαinduced by neuraminidase (NA) treatment. Control represents untreatedsamples.

FIG. 21 is a graph showing that addition of DANA completely rescues thein vivo recovery and survival of mouse platelets treated withneuraminidase. Control depicts the survival of non-treated fresh roomtemperature platelets.

FIG. 22 is a graph showing that platelet GPIbα and GPV receptor lossduring storage at room temperature is inhibited by the addition of DANA.

FIG. 23 is a bar graph depicting the effect of neuraminidase treatmenton β-galactose exposure in the presence of 100 μM metalloproteinase (MP)inhibitor GM6001. β-Galactose exposure was measured byfluorescently-labeled RCA-1 lectin binding.

FIG. 24 is a bar graph depicting the effect of neuraminidase treatmenton platelet GPIbα and GPV receptor surface expression in the presence of100 μM metalloproteinase (MP) inhibitor GM6001. The receptor expressionon MP inhibitor-neuraminidase was set to 100%.

FIG. 25 is a bar graph depicting the effects of recombinant TACE(ADAM17) (TACE) and recombinant TACE and DANA (TACE+DANA) on plateletGPIbα and GPV receptor surface expression. The fact that inhibition ofsialic acid loss prevents receptor cleavage by the metalloproteinaseTACE shows that sialic acid has to be hydrolyzed from glycoproteinsbefore the proteolysis of GPIbα and GPV. The receptors GPIX and αIIbβ3were not affected by treatment with recombinant TACE (not shown).

FIG. 26 is a bar graph depicting the quantification of free sialic acid(FSA) in fresh platelet samples and stored samples at 4° C. and RT forthe indicated time points. FSA concentrations are also shown on the topof each bar graph. Note that FSA detected in RT-stored platelet sampleswas much higher when compared to samples stored at 4° C. for equivalenttime periods.

FIG. 27 are photographs showing the time required to detect bacteria inplatelet samples (TOCD: Time of color detection) stored at 4° C. or atRT in the presence or absence of the sialidase inhibitor, DANA. Thebacterial concentration in the test sample is inversely proportional tothe onset time of color development, i.e., shorter time of colordetection ˜ higher concentration of bacteria; longer time colordetection ˜ lower concentration of bacteria. Selected pictures for theanalysis of Day 9 samples are shown (panels A, B, and C). Bacteria weredetected using an assay technology as described in Example 6. Panel D isa bar graph showing the quantification of the bacterial analysis inplatelet samples stored at 4° C. or RT in the presence or absence ofsialidase inhibitor DANA. TOCD (min) was plotted against the plateletsamples. Note that the time required for in RT stored samples with DANAis equivalent to 4° C. stored samples, indicating that DANA inhibitsbacterial growth as effectively as 4° C.-storage.

FIG. 28 is a line graph depicting the survival of mouse platelets storedfor 48 h by refrigeration in the absence (48 h) or presence of 1 mM DANA(48 h+DANA) in the storage solution. The survival of fresh, isolatedplatelets (RT) is shown for comparison, n=7 for each survival graph.

FIG. 29 is a flow cytometry analysis of fresh platelet (Fresh platelets)size and density (A) and the combined effect of DANA, sialylactose, andglucose on stabilizing RT-stored mouse platelet integrity, as judged bytheir size (FSC) and density (SSC). Analysis of mouse platelets storedfor 48 h at RT in the absence (−preservatives) (B) and presence(+preservatives) (C) of sialylactose, glucose, and DANA is shown. Thecorresponding platelet numbers are shown below the dot plots. Theconcentration of the preservatives is also shown.

FIGS. 30 (A) (B) (C) & (D) is a flow cytometry dot plot analysis ofmouse platelets stored at RT for 48 h in the absence (0 mM DANA; shownin panel (A) or presence of DANA at the indicated concentrations (0.1,1.0, 10.0 mM DANA as shown in panels (B), (C), and (D), respectively).Note that 0.1 mM DANA efficiently preserved the size and density ofplatelets as judged by dot plot analysis. The dot plots are shown in thetop panels. Corresponding flow cytometry histograms of platelet countsand beads (reference) are also shown (lower panels).

FIG. 31 shows bar graphs depicting the cell density of S. marcescensgrown for 48 h in different media with or without 1 mM DANA in the wellsof 96-well PVC plate (panel (A)). FIG. 31 in panel B depicts biofilmformation of S. marcescens, incubated for 48 h in different media withor without 1 mM DANA in the wells of 96-well PVC plate. Also shown inpanel (B), the biofilm in each well was stained with crystal violet, andthe dye was recovered and measured at 595 nm. The absorption at 595 nm(A595 nm) is proportional to the bacterial cells in the biofilm.

FIG. 32 is a bar graph showing the differences in terminal β-galactosecontent on fresh platelets isolated from healthy subjects. Plateletsurface terminal galactose exposure was measured by flow cytometry usingthe β-galactose specific lectin ECL, as depicted in the schematicdrawing of lectin binding to a glycan-structure.

FIG. 33(Aa), (Ab),(Ac), & (Ad) and FIG. 33(B) are a flow cytometry dotplot analysis and corresponding flow cytometry histograms (depicted inFIG. 33 (Aa), (Ab),(Ac), and (Ad)) of mouse platelets stored in 30%plasma and 70% Platelet Additive Solution (PAS) (referred to asINTERSOL® solution) by volume at RT for 48 h in the absence of additive(INTERSOL® solution) (depicted in (Aa)), the presence of 1 mM DANA(INTERSOL® solution+DANA) (depicted in (Ab)), 10 mM glucose (INTERSOL®solution+Glucose) (depicted in (Ac)), and 1 mM DANA plus 10 mM glucose(INTERSOL® solution+DANA+Glucose) (depicted in (Ad)). Note, that theplatelet population appears resting, as judged by their forward and sidescatter characteristics. FIG. 33 (B) is a bar graph showing the percentof acquired events in the gated platelet population for the INTERSOL®solution (depicted in (Ba)), INTERSOL® solution with DANA (depicted in(Bb)), INTERSOL® solution with glucose (depicted in (Bc)), or INTERSOL®solution with both glucose and DANA (depicted in (Bd)).

FIG. 34 is a representative flow cytometry dot plot analysis ofplatelets stored in the absence ((−) DANA) or presence of 0.5 mM DANA((+) DANA) (upper panel (A)). A corresponding histogram of plateletcounts vs side scatter (SSC) is also shown (lower panel (B)). The tablerepresents the mean fluorescence intensity (MFI) measured in the sidescatter (SSC-H (MFI)) in the absence or presence of DANA.

FIG. 35, panel (A), is a representative flow cytometry histogramanalysis of surface P-selectin exposure after human platelet storage inplasma in the absence or presence of DANA as described in FIG. 34.P-selectin exposure was measured using a monocolonal FITC conjugatedantibody to P-selectin (CD62P-FITC). FIG. 35, panel (B), showsquantification of P-selectin positive platelets defined in M2 (asindicated in FIG. 35, panel (A)) and the corresponding MFI.

FIG. 36 is a flow cytometry dot plot analysis of human platelets storedat RT for 7 days in 30% plasma and 70% PAS solution (by volume) (PASa,7.15 mM Na₂HPO₄, 2.24 mM NaH₂PO₄, 10 mM sodium citrate, 30 mM sodiumacetate, 79.2 mM NaCl, 5.0 mM KCl, and 1.5 mM MgCl₂, pH 7.2) in thepresence of 0 (A), 0.1 (B) and 0.5 (C) mM DANA. The platelets aredefined in ‘G1’ while the platelet microparticles are defined in ‘G2’.The gate statistics are shown for each dot plot.

FIG. 37 is a schematic showing sialidase and β-galactosidase activityand a platelet clearance mechanism.

FIG. 38 is a schematic showing Platelet surface β-galactose exposuredetermined by lectin binding. Platelets were isolated from healthyvolunteers and terminal β-galactose exposure was determined by flowcytometry using 1 μg/mL FITC-conjugated RCA-1 lectin. The schemeindicates RCA-1 lectin binding to terminal galactose. Isolated plateletsfrom healthy volunteers differ in terminal β-galactose content and thiscorrelates with platelet ingestion by HepG2 cells in vitro.

FIG. 39A is a graph showing the correlation of HepG2 cells' ingestion ofhuman platelets with β-galactose exposure, by showing the quantificationof platelets recovered from HepG2 cell incubation media. Isolated humanplatelets were labeled with CM-Orange, added to HepG2 cells andincubated for 30 min at 37° C. The number of platelets counted beforeaddition to HepG2 cells was set to 100% for each individual.

FIG. 39B is a bar graph showing the ingestion of fluorescently(CM-orange) labeled fresh platelets, as detected using flow cytometry asan increase in hepatocyte associated orange fluorescence.

FIG. 40 is a line graph showing platelet surface terminal β-galactosechanges during platelet storage. Platelets were isolated from plateletconcentrates (Blood Transfusion Service, Massachusetts General Hospital)at the indicated time points and terminal β-galactose exposure wasdetermined by flow cytometry using 1 μg/mL FITC-conjugated RCA-1 lectin.Platelet concentrates were obtained from the Blood Transfusion Service,Massachusetts General Hospital, Boston, Mass., and stored at roomtemperature under standard blood banking conditions. Platelets wereobtained and analyzed at the indicated time points. Terminal β-galactosecontent decreases on isolated platelet surfaces during platelet storageand correlates with ingestion by HepG2 cells.

FIG. 41A is a line graph showing that the HepG2 cells ingestion of humanplatelets correlates with the decrease in sialic acid and β-galactoseexposure. Quantification of platelets recovered from HepG2 cellincubation media is shown. Isolated human platelets were labeled withCM-Orange, added to HepG2 cells and incubated for 30 min at 37° C. Thenumber of platelets counted before addition to HepG2 cells was set to100% for each individual.

FIG. 41B is a line graph showing the ingestion of fluorescently labeledstored platelets, as detected using flow cytometry, as an increase inhepatocyte associated orange fluorescence.

FIG. 42 is a bar graph showing the analysis of platelet surfacesialidase activity. The enzyme activity was determined using afluorometric assay by incubating the platelets isolated from plateletconcentrates (Bag A or B), with 4-MU-NeuAc. The product 4-MU can bequantified by 355Ex/460Em at pH>10. The donors exhibited variableplatelet surface sialidase activity at the early stage of the storage(Day 1), which became up-regulated after further storage (Day 6). DonorB has higher activity than Donor A on both Day 1 and Day 6. Sialidaseactivity on platelet surface increases during room temperature storage.

FIG. 43 is a bar graph showing the analysis of platelet surfaceβ-galactosidase activity. The enzyme activity was determined using acolorimetric assay by incubation of platelets (Bag A or B) withGalβ-pNP. The product pNP can be read at 405 nm at pH>10. Donors A and Bexhibited variable platelet surface β-galactosidase activity at theearly stage of the storage (Day 1), which became up-regulated afterfurther storage (Day 6). Donor B has higher activity than Donor A onboth Day 1 and Day 6. β-Galactosidase activity on platelet surfaceincreases during room temperature storage.

FIG. 44 is a bar graph showing that THP-2 cells ingestion of humanplatelets correlates with decrease in β-galactose exposure. Isolatedhuman platelets were labeled with CM-Orange, added to THP-1 cells andincubated for 30 min at 37° C. Ingestion of fluorescently labeledcontrol fresh room temperature platelets and platelets treated byβ-galactosidase was detected using flow cytometry, as an increase inhepatocyte associated orange fluorescence.

FIG. 45A is a line graph of platelet surface exposure ofphosphatidylserine (PS) as measured by FITC labeled Annexin V bindingover the time of platelet storage (n=4).

FIG. 45B is a bar graph of platelet surface exposure of PS as measuredby FITC labeled Annexin V binding after storage for 7 days (n=4).

FIG. 46A is a line graph of platelet surface exposure of PS as measuredby FITC labeled Annexin V binding over the time of platelet storage(n=4).

FIG. 46B is a bar graph of platelet surface exposure of P-selectin asmeasured by FITC labeled CD62P antibodies binding after storage for 9days (n=4) (p<0.01).

FIG. 47 is a scatter plot showing the percentage of fresh and storedplatelets recovered at 5 min following transfusion. Platelets werestored for 20 hours at room temperature in plasma, VPAS, or VPAS+2.Fresh non-stored platelets are used as control (n=3 for each group).

FIG. 48 is a line plot showing short-term survival of fresh and storedplatelets following transfusion. Platelets were stored for 20 hours atroom temperature in plasma, VPAS/Plasma (70:30), or VPAS+/Plasma(70:30). Fresh non-stored platelets are used as control (n=3 for eachgroup).

FIG. 49 is a line graph showing the percent (%) of fluorescent plateletsurvival following transfusion over time (between 0 and 72 hours) offresh platelets, plasma platelets, pPAS Platelets (which is as “PPS9”without the DGJ described Table 3) and pPAS+DGJ Platelets (referred toas “PPS9” in Table 3).

DETAILED DESCRIPTION OF THE INVENTION

A description of preferred embodiments of the invention follows.

Platelet Protection Solution (PPS)

After platelets are obtained from a donor, they can be suspended influid referred to as Platelet Protection Solution (PPS). Essentially,PPS replaces a portion of the plasma in which the isolated platelets areplaced during apheresis. PPS is a medium that is generally aphysiologically compatible, aqueous electrolyte solution. In addition tocertain agents that can be normally present in such solutions in varyingcombinations and concentrations as described hereinafter, the PPSsolution of the present invention includes one or more β-galactosidaseinhibitors with or without one or more sialidase inhibitors, andoptionally one or more glycan modifying agents.

PPS solutions are used because they are believed to reduce allergic andfebrile transfusion reactions, facilitate ABO-incompatible platelettransfusions, optimize the use of pathogen inactivation techniques andmake more plasma available for other purposes (e.g., for fractionation).

One embodiment of the present invention includes a PPS solution havingthe β-galactosidase inhibitor, and optionally a glycan-modifying agent.Another embodiment of the present invention includes a PPS solutionhaving the β-galactosidase inhibitor, the sialidase inhibitor andoptionally a glycan-modifying agent. More specifically, the presentinvention includes a PPS composition having a β-galactosidase inhibitorwith or without a sialidase inhibitor, and/or a glycan-modifyingcomposition, and one or more of PPS components (e.g., salts, buffers,nutrients, or any combination thereof). PPS of the present invention caninclude a variety of components such as one or more salts (e.g., NaCl,KCl, CaCl₂, MgCl₂, and MgSO₄), one or more buffers (e.g., acetate,bicarbonate, citrate, or phosphate) and nutrients (e.g., acetate,gluconate, glucose, maltose, or mannitol).

The term “Platelet Protection Solution” or “PPS” of the presentinvention refers to the solution or medium having at least one or moreβ-galactosidase inhibitors, or both one or more β-galactosidaseinhibitors and one or more sialidase inhibitors; and one or more storagemedium components and, optionally, one or more glycan modifying agents.The “inventive composition” includes one or more β-galactosidaseinhibitors, or both one or more β-galactosidase inhibitors and one ormore sialidase inhibitors and, optionally, one or more glycan modifyingagents. The phrase “platelet composition” or “platelet storagecomposition” refers to the resulting storage composition (prior totransfusion into a recipient), which includes the PPS of the presentinvention, the platelets, and optionally, any plasma and/oranticoagulant associated with the platelets.

Additionally, the medium of the PPS of the present invention includes aphysiologically compatible, aqueous electrolytic solution. Suchsolutions can contain ionic elements in solution such as sources ofsodium, potassium, magnesium, calcium, chloride and phosphate. The PPSof the present invention can also contain, e.g., sources of citrate thatcan be added in the form of citric acid or sodium salt. The solution ofthe present invention further includes, for example, carbon or nutrientsource, such as acetate, glucose or gluconate, and can be present incombination with a salt. A phosphate source, in an embodiment, can beincluded to help maintain ATP production. These elements can be presentin the solution of the present invention in an amount ranging from about0.1 mM to about 450 mM (e.g., 10, 20, 30, 40, 50, 60, 70, 80, 90, 100,150, 200, 250, 300, 350, 400, 450 mM). In an embodiment, certaincomponents that are not important in protecting platelets during storagecan be omitted. For example, in certain embodiments, calcium or glucoseare not included in formulations, such as PPS 5. See Tables 1 and 3. Thesolution is maintained at a pH ranging from about 6.4 and about 7.6(e.g., about 7.1 to about 7.4), and preferably at pH of about 7.2.

In an embodiment, a source of sodium (Na) can be present in the PPS ofthe present invention in an amount between about 100 and 300 mM (e.g.,between about 150 mM and about 250 mM). In a particular embodiment, asource of sodium is present at about 190 mM. Sodium can be present as asalt or in combination as a buffer, or carbon source. For example,sodium can be present in the form of sodium chloride (NaCl), sodiumcitrate, sodium acetate, sodium phosphate or a combination thereof.Other suitable sources of sodium can be used in the PPS of the presentinvention including those known in the art or later discovered.

A source of chloride (Cl) can also be present in the PPS of the presentinvention in an amount between about 40 mM and about 110 mM (e.g.,between about 60 mM and about 100 mM). In one embodiment, chloride ispresent in an amount between about 100 mM and about 110 mM, and inparticular between about 105-106 mM. For example, in the PPS5formulation of Table 3, it is present in about 105.3 mM. In the PPS 1formulation, the source of chloride is present at about 87.2 mM.Chloride can be present in the form of sodium chloride (NaCl), magnesiumchloride (MgCl₂), potassium chloride (KCl), or a combination thereof.Any source of chloride known in the art or later discovered can be usedwith the present invention so long as it is suitable for use with PPS ofthe present invention. Na⁺ and Cl⁻, mainly in the form of NaCl, aretonicity modifiers that contribute to the isotonicity of plateletprotection solution.

A source of potassium, in an embodiment, can be present in the PPS ofthe present invention. It can be present in an amount ranging betweenabout 0.5 mM and about 10 mM, and for example, between about 3 mM andabout 8 mM. In a particular embodiment, potassium is present in anamount of about 5 mM. Potassium sources include potassium chloride,potassium citrate, potassium acetate, potassium phosphate, potassiumsulfate, or a combination thereof. Other sources of potassium known inthe art or later discovered can be used with the present invention. Thepresence of potassium ion in the medium can assist, in certain aspects,in maintaining intracellular magnesium ion concentration. Potassium ioncould also be involved in the transport of pyruvate across themitochondria membrane for oxidative phosphorylation in the citric acidcycle (TCA cycle). In addition, K⁺ plays important roles in membranestability by contributing to the electrical continuity of lipids andproteins.

Magnesium is another salt that can be included in the PPS of the presentinvention. A source of magnesium can be present in an amount rangingbetween about 0.5 mM and about 5.0 mM, and in particular, in an amountranging between about 1 mM and 2 mM. In an embodiment, magnesium ispresent in the PPS of the present invention at about 1.5 mM. Sources ofmagnesium include magnesium chloride, magnesium citrate, magnesiumsulfate, and a combination thereof. Sources of magnesium known in theart or later discovered can be used. In one embodiment, magnesium ioncan be present in the PPS of the present invention at concentrationsclose to plasma levels, which will be about 3 mEq/L (1.5 mM). Mg²⁺ mightbe necessary to maintain membrane ATPase activity. In an aspect,magnesium ion in the medium should maintain the optimal intercellularmagnesium levels in the platelets and may promote oxidativephosphorylation in the platelets and in so doing help maintain the pH ofthe medium. Furthermore, Mg²⁺ plays important roles in membranestability by contributing to the electrical continuity of lipids andproteins.

Calcium is another yet salt that can be included in the PPS of thepresent invention. A source of calcium can be present in an amountranging between about 0.0 mM and about 2.5 mM (e.g., between about 1 mMand 2 mM). In a certain embodiment, calcium is present in the PPS of thepresent invention in about 1.5 mM. In another embodiment, calcium is notpresent at all. See Tables 1 and 3. Sources of calcium include calciumchloride, calcium acetate, calcium citrate, or a combination thereof.Sources of calcium known in the art or later discovered can be used.

Citrate can be used to buffer the solution. A source of citrate ispresent in the PPS of the present invention in an amount ranging betweenabout 2 mM and about 20 mM, and for example, in an amount between about5 mM and about 15 mM. In an aspect, the PPS of the present inventionincludes about 10 mM of citrate. Examples of citrate sources that can beused in the present invention include sodium citrate (e.g., monosodiumcitrate, disodium citrate, trisodium citrate), citric acid, potassiumcitrate, magnesium citrate and a combination thereof. Other sources ofcitrate can be used including those known in the art or later discoveredso long as it is suitable for use with PPS of the present invention.Citrate plays multiple roles in PPS of the present invention as ananticoagulant, a carbon source for the TCA cycle and buffer.

Acetate is yet another component of the PPS of the present invention.Acetate is a carbon source used as a nutrient for the isolatedplatelets. A source of acetate can be present in an amount rangingbetween about 10 mM and about 50 mM, and for example, in an amountranging between about 25 mM and about 45 mM. The PPS of the presentinvention includes about 30 mM of acetate. Sources of acetate includesodium acetate, potassium acetate, magnesium acetate or a combinationthereof. Other sources of acetate can be used including those known inthe art or later discovered so long as it is suitable for use with PPSof the present invention. Acetate serves as carbon and buffer.

In the PPS of the present invention, a nutrient source can be provided.Acetate and other carbohydrates such as glucose or sucrose, as well ascitrate, can be used individually or in various combinations to providea source of energy for platelets in storage by being a source ofintermediate metabolites for the production of energy in the citric acidcycle. A combination of a carbon source can be used. In the case thatglucose and/or sucrose is used, the concentration can be present in anamount ranging from about 0.1 mM to about 30 mM (e.g., about 2 mM toabout 22 mM). In an embodiment, glucose can be omitted from theformulation. See Tables 1 and 3.

Other nutrients can be substituted for or included with the acetate ofthe PPS of the present invention. For example, oxaloacetate can bepresent in the PPS of the present invention or can be added to plateletsuspension after the PPS of the present invention has been added to aplatelet rich fraction. Oxaloacetate is a four-carbon molecule found inthe mitochondria that condenses with Acetyl Co-A to form the firstreaction of the TCA cycle (citric acid cycle). Oxaloacetate can besupplied to the stored platelets either directly or in the form ofprecursor amino acids such as aspartate. In some embodiments,oxaloacetate can be present in the PPS of the present invention fromabout 10 mM to about 45 mM. More particularly, oxaloacetate can bepresent in the PPS of the present invention from about 20 mM to about 40mM, or from about 24 mM to about 36 mM, or from about 28 mM to about 33mM.

Phosphate (PO₄) is another component that can be used in the PPS of thepresent invention. A source of phosphate can be present in the PPS ofthe present invention in an amount ranging between about 5 mM and about50 mM (e.g., between about 20 and 40 mM). In a particular embodiment, asource of phosphate is present in about 28 mM. Forms of phosphateinclude sodium monophosphate, diphosphate, triphosphate, or acombination thereof. Other sources of phosphate known in the art ordiscovered in the future can be used.

Components such as acetate, citrate and phosphate can be added incombination with one or more salts, such as the calcium, magnesium,potassium, or sodium salts or any sub-combination of these salts tobalance the osmolarity of the buffered solution.

In an embodiment, the PPS of the present invention includes one or moreβ-galactosidase inhibitors with or without one or more sialidaseinhibitors, and optionally, one or more glycan modifying agents, and thecomponents described in Table 1:

TABLE 1 Range (mM) 5 Low High PPS1a PPS2a PPS3a PPS4a PPS5 PPS6 PPS7PPS8 PPS9 Sodium [Na] 100 300 156.7 148.3 155.2 146.8 177.7 169.2 176.7168.2 147.3 Chloride [Cl] 40 110 87.2 78.8 87.7 79.3 105.3 96.8 103.394.8 80.8 Citrate 2 20 10 10 10 10 10.8 10.8 10.8 10.8 10.0 Acetate 1050 30 30 30 30 32.5 32.5 32.5 32.5 30.0 Phosphate [PO₄] 5 50 9.4 9.4 9.49.4 9.4 9.4 9.4 9.4 9.4 Potassium [K] 0.5 10 5.0 5.0 5.0 5.0 5.0 5.0 5.05.0 5.0 Magnesium [Mg] 0.5 5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 Calcium[Ca] 0 2.5 0 0 1.0 1.0 0 0 0.0 0.0 0.0 [Glucose] 0 30 0 16.8 0 16.8 0 170.0 17.0 16.8 [DANA] 0 10.0 1.0 1.0 1.0 1.0 1.0 1.0 0.0 0.0 0.0 1- 010.0 1.0 1.0 1.0 1.0 2.0 2.0 0.0 0.0 2.0 Deoxygalactonojirimycin (DGJ)Total (mM) 301.8 301.8 301.8 301.8 345.2 345.2 339.2 339.2 302.8

The PPS of the present invention as described herein can also bebuffered, in an embodiment, by amino acids. The amino acids can be usedas the primary buffering agents, or can be used in conjunction withother buffering agents such as phosphate. In one embodiment the aminoacid, histidine, can be used to buffer the storage solution. Thus, thestorage solution can contain amino acids from about 1 mM to about 7 mM,or from about 2 mM to about 5 mM.

In addition to or as an alternative to the foregoing, the PPS disclosedherein can further include other components that promote oxidativephosphorylation. An antioxidant can be added to the PPS or plateletcomposition of the present invention. Examples of antioxidants includeglutathione, selenium, and the like. In some embodiments the antioxidantcan be present in the PPS of the present invention in an amount rangingbetween about 0.5 μM to about 3 mM (e.g., about 1.0 μM to about 2 mM).In some embodiments, glutathione, or its precursor N-acetylcysteine,and/or selenium alone or in combination can be present in the PPS in anamount between about 0.5 μM to about 3 mM (e.g., about 1.0 μM to about 2mM).

To further promote oxidative phosphorylation, the PPS of the presentinvention can further include components that assist in stabilizingmembranes. For example, a phospholipid or a mixture or phospholipids canbe included in the storage solution. In some embodiments, phospholipidscan be present in the PPS of the present invention in an amount rangingfrom about 0.1 mg/mL to about 7.5 mg/mL (e.g., between about 0.25 mg/mLto about 5 mg/mL). More particularly, L-alpha phosphatidylcholine can bepresent in the PPS of the present invention in an amount between about0.1 mg/mL to about 7.5 mg/mL (e.g., about 0.25 mg/mL to about 5 mg/mL).

Additional components that can be included in the PPS of the presentinvention are non-essential amino acids. For example, non-essentialamino acids in an amount ranging from about 0.5 mM to about 14 mM can bepresent in the PPS (e.g., about 1.0 mM to about 10 mM). In anembodiment, L-alanine can be included in an amount ranging from about0.5 mM to about 14 mM (e.g., about 1.0 mM to about 10 mM).

Unsaturated free long chain fatty acids can further be included in thePPS of the present invention. The PPS described herein can contain anamount of unsaturated free long chain fatty acids in a range betweenabout 0.05 mM and about 1.5 mM (e.g., about 0.1 mM to about 1 mM). In anembodiment, the PPS of the present invention can contain palmitic acidfrom about 0.05 mM to about 1.5 mM, or about 0.1 mM to about 1 mM.

United States Pharmacopeia (USP) water for injection (WFI) can be usedas a solvent to make the buffer solution for the PPS of the presentinvention.

The phrase “platelet composition” (e.g., the PPS of the presentinvention and isolated platelets) refers to a composition whose totalvolume contains between about 0% to about 50% by volume of plasma. Theplatelet composition, in one aspect, contains less than about 50% (e.g.,less than about 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1%) by volume plasma. Conversely, the platelet storagecomposition of the present invention has between about 50% and about 99%(e.g., about 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%) by volume ofPPS of the present invention, which contains one or more β-galactosidaseinhibitors with or without one or more sialidase inhibitors, in anelectrolytic solution, and also phosphate and/or buffering compounds,carbon source (s), and optionally, one or more glycan modifying agents.In certain embodiments, the platelet storage composition is essentiallyplasma free having mostly the PPS of the present invention andplatelets. In an embodiment, the platelets generally make up about 1% byvolume of the total platelet composition.

In an embodiment, once the PPS of the present invention is added to theisolated platelets, PPS of the present invention constitutes about 70%and the plasma constitutes about 30% of the isolated platelet solution.The percentage of PPS of the present invention by volume can varydepending on its use, e.g., for transfusion into chronically anemicpatients or acutely anemic patients. Hypervolemia is a concernespecially in trauma patients suffering from acute anemia. Accordingly,the percentage of PPS can be modified to minimize or avoid hypervolemia.

The platelet composition in the PPS of the present invention can beassessed at one or more time points during storage. Assessment of theplatelet content, platelet morphology, metabolism, bacterialproliferation, the extent of platelet activation, extent of lysis, or acombination thereof can be performed. Additionally, the amount ofcleaved sialic acid or the amount of β-galactose exposed on the glycanmolecules on the platelet surface can be determined as a measure of theplatelet's likelihood to be cleared from circulation. The assessment ofplatelets, their function and bacterial proliferation is furtherdescribed herein to assess the platelets' ability to be transplanted,survive in vivo and maintain hemostasis after transfusion. The PPS ofthe present invention allows platelets to be stored longer, and havelonger circulation and maintain hemostasis after transfusion, ascompared to platelets not stored in the PPS of the present invention.Storage times, circulation times and hemostasis are also furtherdescribed herein.

Metabolism of platelets can be assessed by measuring ATP and levels ofglucose, lactate and lactate dehydrogenase (LDH). ATP measurements canbe carried out using assays known in the art such as Bioluminescentassay kit (Sigma, Poole, Dorset, UK). Glucose, lactate, and LDH can alsobe measured using assays known in the art, such as Vitros DT60 11chemistry system (Shield, Kimbolton, Cambridgeshire, UK). The plateletsuse a carbon source such as acetate during metabolism to maintain ATP, amajor energy carrier. The PPS of the present invention can maintain a pHof between about 6.4 and about 7.6, and preferably between about 7.1 toabout 7.4.

Applicants have characterized several underlying mechanisms that accountfor the high susceptibility of platelets to irreversible intolerance bythe recipients of transfusions and the resulting loss of platelet's invivo hemostatic activity. Applicants' discoveries are related to sialicacid and its role in the viability of platelets.

Surprisingly, Applicants have found that the catalytic hydrolysis ofsialic acid residues from platelet surface glycans by the platelet's ownsialidase enzymes generally contributes to the irreversible intoleranceof platelets. Applicants have further discovered that β-galactosidaseenzyme surface activity actually increases during platelet storage.Additionally, the Applicants discovered that endogenous sialidaseactivity increases during platelet storage. Yet another surprisingdiscovery is that sialidase-producing bacteria desialylate plasma andplatelet sialioglycoconjugates to obtain nutrients such as sialic acidwhich supports bacterial growth and proliferation. See FIG. 1A.Bacterial proliferation leads to biofilm formation, platelet activationand aggregation. Desialylated platelets enhance bacteria-plateletinteraction and eventually are cleared from circulation vialectin-mediated mechanism (FIG. 1B). Accordingly, the addition of asialidase inhibitor prevents sialic acid from being cleaved from theplatelet surface, thereby preventing platelet clearance and prolongingits survival. Also, addition of a β-galactosidase inhibitor preventsβ-galactose from being cleaved from the platelet surface, which alsohelps to prevent platelet clearance and increase its in vivo survival.Additionally, a sialidase inhibitor inhibits the proliferation ofbacteria in a platelet preparation (FIG. 1C). The dual sialidaseinhibitor-function provides a superior platelet preparation with longersurvivals and reduces the chance of causing bacteria-related sepsis whentransfused into a recipient at the point of care.

With these counterintuitive and surprising results in hand, Applicantshave developed methods to effectively treat platelets with inhibitors ofsialidase after they are harvested from donors and prior to storage ator below room temperature. Treated with β-galactosidase inhibitors, orwith sialidase inhibitors and β-galactosidase inhibitors, the inventiveplatelet compositions retain in vivo hemostatic activity for longerdurations as compared to untreated platelets. The inventive plateletcompositions treated with β-galactosidase inhibitors with or withoutsialidase inhibitors can be stored for prolonged periods at or belowroom temperature as compared to untreated platelets. The storage ofplatelets according to the inventive methods extends the shelf life ofplatelets and helps increase the supply of platelets that remain viablefor transfusion with inhibited bacterial proliferation.

As noted, Applicants' discoveries are related to sialic acid andβ-galactose and their role in the viability of platelets. The hydrolysisof sialic acid from the outer membrane of platelets is believed tocontribute to the unique and irreversible in vivo intolerance ofplatelets. Studies have reported that platelets lose sialic acid frommembrane glycoproteins during aging and circulation, and that in vitrodesialylated platelets are cleared rapidly. Loss of sialic acid exposesunderlying immature glycans such as β-galactose. Asialoglycoprotein(ASGP) receptors are known to mediate endocytosis of proteins, cells andparticles carrying exposed β-galactose. Many cells, including hepaticmacrophages and hepatocytes, express and present the ASGP receptor.Accordingly, it is believed that when endogenous sialidase enzymescleave sialic acid residues from the platelet surface, penultimatesugars such as β-galactose are exposed on the platelet surface andplatelets undergo ASGP-mediated ingestion after transfusion. Similarly,it is also believed that when β-galactose is cleaved andN-acetylglucosamine (GlcNAc) is exposed on the platelet surface, theplatelets with GlcNAc exposed are also cleared. It is also believed thatGlcNAc removal exposes mannose, which can be readily recognized bymacrophage mannose receptors, triggering immediate platelet clearance.

While the loss of surface receptors (e.g., GPIb and GPV) on plateletshas been associated with platelet survival, prior to the presentinvention the role of surface sialic acid and/or β-galactose withrespect to surface receptors on platelets was unknown. Furthermore, therole of surface sialic acid and/or β-galactose regarding the survival ofplatelets was unclear. Applicants have used in vitro and in vivo studiesto characterize relationships between surface sialic acid/β-galactose,and platelet receptor loss. Accordingly, Applicants' results have beenapplied to the inventive methods described herein for prolonging thesurvival of platelets. This relationship between surface sialicacid/β-galactose and platelet receptor loss turns out to be an importantfactor in determining platelet survival. Applicants have found thatinhibiting the loss of surface sialic acid and/or β-galactose preventsplatelet surface receptor GPIb and GPV loss during storage in vitro andrescues platelet survival in vivo.

For example, mouse platelets stored at room temperature for 6 h lostsurface sialic acid, as evidenced by flow cytometry data providedherein. See Exemplification. This loss correlated with a 30-60% loss ofsurface receptors GPIb and GPV, but not GPIX and integrin αIIbβ3.Furthermore, treatment of mouse platelets with the neuraminidase (NA)substrate, fetuin, partially decreases the loss of GPIb and GPV to10-20%. In vitro, sialic acid was cleaved from the platelet surface byadding α2-3,6,8-neuraminidase (NA; Vibrio cholerae) or α2-3,6,-NA(Clostridium perfringens) to mouse platelets. Removal of sialic acidcorrelated with the removal of 50-60% of surface GPIbα and GPV, but notGPIX and integrin αIIbβ3. Addition of fetuin, or the more specificsialidase inhibitor, the sodium salt of2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA), completely preventedthis loss, as determined by both flow cytometry and Western blotanalysis, also provided herein.

The data described herein also show that human platelets have variablesurface sialidase and β-galactosidase activities among donors, and showthat both are up-regulated during platelet storage at room temperature(RT). The data also show that human platelets have variable surfaceβ-galactose exposure/sialic acid loss among individual donors. Duringstorage at RT, platelet surface β-galactose exposure appears to peak atday 2, then decrease during further storage. Platelet surfaceβ-galactose content correlates positively with ingestion by HepG2 cells,and crosstalk with platelet surface glycosidase activities. Since theassociation with β-galactosidase goes along with Neu1 sialidaseactivity, the concerted up-regulation of sialidase and β-galactosidaseactivities on platelet surface indicates that the multi-enzyme complexis relocated from lysosome to platelet surface during plateletstorage/aging, possibly through the fusion between platelet membrane andlysosomal membrane. See FIG. 37. The relocation of both Neu1 andβ-galactosidase onto platelet surface catalyzes the sequentialdegradation of platelet surface glycans, loss of sialic acid, followedby β-galactose, exposing terminal N-acetylglucosamine (GlcNAc). GlcNAccan be further removed, exposing the mannose residues. Mannose can bereadily recognized by macrophage mannose receptors, triggering immediateplatelet clearance. Accordingly, inhibiting β-galactosidase activityprolongs the platelet storage and increases in vivo survival ofplatelets. Also, by inhibiting both sialidase enzyme and β-galactosidaseactivity, it is possible to prolong the platelet storage and increase invivo survival of platelets.

The clearance of platelets is exacerbated upon cooling. It has beendiscovered that cooling of human platelets causes clustering of the vonWillebrand factor (vWf) receptor complex a subunit (GPIbα) complexes onthe platelet surface. The clustering of (GPIbα) complexes on theplatelet surface elicits recognition by macrophage complement type threereceptors (αMβ2, CR3) in vitro and in vivo. CR3 receptors recognizeN-linked sugars with terminal β-GlcNAc on the surface of platelets,which have formed GPIbα complexes, and phagocytose the platelets,clearing them from the circulation and resulting in a concomitant lossof hemostatic function. Although capping the β-GlcNAc moieties bygalactosylation prevents clearance of short-term-cooled platelets, thisstrategy is ineffective after prolonged refrigeration (e.g.,refrigeration of platelets longer than 5 days). Prolonged refrigerationfurther increased the density and concentration of exposed galactoseresidues on platelets GPIbα such that hepatocytes, throughAshwell-Morell receptor (ASGP receptor or hepatic lectin) binding,become increasingly involved in platelet removal. Macrophages rapidlyremoved a large fraction of transfused platelets independent of theirstorage conditions. With prolonged platelet chilling,hepatocyte-dependent clearance further diminishes platelet recovery andsurvival after transfusion. Inhibition of chilled platelet clearance byboth β2 integrin and Ashwell-Morell receptors may afford a potentiallysimple method for storing platelets in the cold.

As noted above, Applicants have discovered that sialidase enzymeactivity is platelet-derived, not plasma-derived, and sialidase enzymeactivity and β-galactosidase enzyme activity substantially increase onthe platelet surface during the storage of platelets. Specifically,Applicants have discovered that human platelets contain the sialidasesNeu1 and Neu3, and release Neu1 into plasma at room temperature, andmore so upon storage in the cold, but it is the surface Neu1 beinginvolved in the removal of surface sialic acid from glycans on thesurface of platelets. Similarly, Applicants have also discovered thatβ-galactosidase is released from the platelet to the platelet surface,along with Neu1, and is involved in the removal of β-galactose from theglycans on the surface of platelets.

The present invention provides platelet compositions and methods forprolonging in vivo hemostatic activity and reducing platelet clearance,wherein the platelets are obtained from a donor and treated with aβ-galactosidase inhibitor or with both a β-galactosidase inhibitor and asialidase inhibitor to counteract the effects of β-galactosidaseactivity or both β-galactosidase activity and endogenous sialidaseactivity, and inhibit bacterial proliferation. Also provided arecompositions and methods for prolonging the storage of viable platelets,such as mammalian platelets, particularly human platelets. The inventionalso provides methods for making improved platelet compositions.

The present invention, in certain aspects, provides plateletcompositions that have enhanced circulation properties and that retainsubstantially normal in vivo hemostatic activity. In certainembodiments, the invention provides a novel platelet compositioncomprising one or more β-galactosidase inhibitors with or without one ormore sialidase inhibitors. As noted, sialidase enzymes catalyze thehydrolysis of terminal sialic acid residues from host cell receptors,and β-galactosidase enzymes catalyze the hydrolysis of β-galactoseresidues from the receptors. Thus, sialidase inhibitors and/orβ-galactosidase inhibitors are used in numerous aspects of the presentinvention to reduce sialidase enzyme activity/β-galactosidase enzymeactivity, prevent the hydrolysis of terminal sialic acid/β-galactoseresidues from platelet surface glycans, inhibit bacterial proliferationand prolong the in vivo hemostatic activity of platelets fortransfusion.

The present invention provides for platelet compositions and relatedmethods to prepare, store, and preserve platelet compositions thatenhance the platelet function and/or allow platelets to retainsubstantially normal in vivo hemostatic activity after platelets havebeen stored at or below room temperature. Certain underlying mechanismshave been discovered that contribute to the high susceptibility ofplatelets to undergo irreversible intolerance or loss of platelet invivo hemostatic activity experienced by recipients of platelettransfusions. The hydrolysis of β-galactose residues from plateletsurface glycans by β-galactosidase enzymes contributes to theirreversible intolerance of platelets. Similarly, the hydrolysis ofsialic acid and β-galactose residues from platelet surface glycans bysialidase and β-galactosidase enzymes, respectively, contributes to theirreversible intolerance of platelets. “Irreversible intolerance” refersto a platelet's inability to retain or return to normal plateletfunction survival after being subjected to temperatures below that ofroom temperature. “Platelet viability” is defined as the platelet'sability to survive in vivo.

The present invention provides platelet compositions and methods ofinhibiting β-galactosidase enzyme activity, or both β-galactosidaseenzyme activity and sialidase enzyme activity; in platelets isolatedfrom a donor and stored at or below room temperature. Thus, in certainaspects, the invention provides compositions having one or moreβ-galactosidase inhibitors with or without one or more sialidaseinhibitors, and optionally one or more glycan-modifying agents. Thepresent invention, in other aspects, provides methods for increasing thecirculation time of platelet compositions having one or moreβ-galactosidase inhibitors, or both one or more β-galactosidaseinhibitors and one or more sialidase inhibitors. The present inventionfurther provides platelet compositions and methods for reducedtemperature storage of platelets, which increases the storage time ofthe platelets, as well as methods for reducing clearance of orincreasing the circulation time of a population of platelets in amammal. Also provided are platelet compositions and methods for thepreservation of platelets with preserved hemostatic activity as well asmethods for making platelet compositions and pharmaceutical compositionsthereof containing the platelet compositions and for administering thepharmaceutical compositions to a mammal to mediate hemostasis. Alsoprovided are kits for treating a platelet preparation for storage andcontainers for storing the same.

The Platelet and how it is Isolated

The term “isolated” as used herein means separated away from its nativeenvironment. As used herein with respect to a population of platelets,isolated refers to removing platelets from the blood of a mammal.

Based on standard blood collection methods, there are generally twotypes of donated platelets: random donor platelets and single donorplatelets. Random donor platelets are platelets isolated from wholeblood donations by means of any one of several standard methodspracticed by those skilled in the art, and two or more random donorplatelets are subsequently pooled in a quantity sufficient to constitutea therapeutic dose prior to transfusion to a patient. A single randomdonor platelet can also be used without pooling for pediatric patients.Current standard methods include isolating random donor platelets from abuffy coat, a platelet button, platelet rich plasma, and the like.Single donor platelets are platelets obtained from one donor by means ofcentrifugal separation in an apheresis machine in a quantity sufficientto constitute one or more therapeutic dose(s) for subsequent transfusionto a patient(s). Apheresis machines used currently for the collection ofsingle donor platelets are manufactured by companies such as Terumo BCT(Terumo Corporation), Fenwal Inc., and Haemonetics Corporation. CurrentAABB (formerly the American Association of Blood Banks) Standards definea therapeutic dose of platelets as approximately ≧3×10¹¹ platelets.

To carry out the methods described herein, either random donor plateletsor single donor platelets are isolated from a donor by means of standardtechniques known to one skilled in the art. The isolated plateletpreparation is treated with one or more β-galactosidase inhibitors withor without sialidase inhibitors and/or glycan-modifying agents asdescribed herein.

Random donor platelets are obtained from whole blood donations. Wholeblood can be obtained from a donor and prepared by a suitable methoddepending on the type of blood components desired. The present inventioninvolves isolating platelets in the form of a buffy coat, a plateletbutton, platelet concentrate, platelet rich plasma, and the like.

In the United States, the collection and processing of all bloodcomponents for transfusion are controlled by FDA regulations and AABBStandards.

Whole blood is comprised of a number of components including plasma, redblood cells, platelets, white blood cells, proteins and othercomponents. Accordingly, in addition to platelets, other components fromwhole blood can be isolated and prepared (e.g., red blood cells, plasma,etc.) when a unit of blood is obtained from a donor. Whole blood isgenerally collected from a donor by venipuncture. The container (e.g.,bag or tube) into which one deposits the blood can contain ananticoagulant such as a citrate or citrate dextrose based component,e.g., citrate phosphate dextrose (CPD or CP2D), citrate phosphatedextrose adenine1 (CPDA-1).

During routine blood collection, a 600 mL bag that contains 70 mL ofanticoagulant is used to collect approximately 500 mL±10% of wholeblood, or 63 mL of anticoagulant is used to collect 450 mL±10% of wholeblood. The whole blood collection bag often has satellite bags attachedthereto to hold isolated components. At the time whole blood iscollected, tubes of donor blood samples are also collected for use inperforming certain required tests on each blood donation, including ABOand Rh determination, infection disease markers testing, and the like.

Platelets are normally separated from whole blood and other bloodcomponents by centrifugation. Centrifuge technology allows separation ofblood components by their various densities. Therefore, the liquid andcellular constituents of whole blood are separated into distinct layersas the result of centrifugation, ranging from red blood cells (RBC), themost dense, to plasma, the least dense. The time of centrifugationvaries depending on the centrifuge and the g-force provided by thecentrifuge. The amount of time of centrifugation can be determined byone of skill in the art. Companies such as Sorvall and Beckmanmanufacture centrifuges that can be used for this process.

Appropriate centrifugation (e.g., a soft spin) results in a bag thatcontains a mass of RBC at its distal end and a mass of platelet richplasma (PRP), a mixture of platelets and plasma at its proximal end,with a meniscus formed primarily by white cells in between the twolayers. By means of the use of a plasma expressor or extractor (made bycompanies such as Fenwal, Inc. and Terumo Corporation), the PRP isexpressed into a satellite bag, leaving the mass of RBC in the originalwhole blood collection bag.

The satellite bag containing the PRP is centrifuged again (e.g., hardspin) to separate the plasma from the platelets. Upon re-centrifugation,the platelets, because of their greater density, form a looselyaggregated cluster called a platelet button. By use of a plasmaexpresser or extractor, the platelet poor plasma (PPP) can then beexpressed into a second satellite bag leaving the platelet button and asmall volume of plasma (together, known as platelet concentrate) in thefirst satellite bag. The platelet concentrate consists of a volume ofapproximately 30 to 70 mL, and the PPP consists of a fluid volume ofapproximately 180 to 320 mL. Each separated blood component, i.e., RBC,PPP or platelet concentrate is known as a “unit”, and each is transfusedseparately.

Generally, the bag of platelet concentrate contains a minimum of 5.5×10⁹platelets. Units of platelet concentrate are stored at 20-24° C. onmechanical rotators. Platelets not treated with the compositions of thepresent invention have a shelf life of about 5 days.

As generally practiced by those skilled in the art, 4-6 plateletconcentrate units are pooled to obtain a single therapeutic dose fortransfusion to a patient. The pooled platelet concentrate has about3.0×10¹¹ platelets or more. The pooled and non-pooled plateletconcentrate obtained from this process comprise one form of “isolatedplatelets” that can be utilized in the present invention or treated withthe inventive compositions described herein. In a particular embodiment,the bag used for pooling the platelet concentrate can have the inventivecompositions described therein (e.g., β-galactosidase inhibitor;β-galactosidase inhibitor and sialidase inhibitor; β-galactosidaseinhibitor and glycan-modifying agent; β-galactosidase inhibitor andsialidase inhibitor and glycan modifying agent), as further describedherein. Alternatively, the inventive composition can be added to theplatelet concentrate before, after, or during pooling.

Random donor platelets may also be isolated by the “buffy coat” methodgenerally used in Europe and Canada. Whole blood is obtained, asdescribed herein, and undergoes a hard spin centrifugation. The hardspin results in a bag having plasma as the top fraction, red blood cellsas the bottom fraction, and a middle layer containing platelets andleukocytes. This middle layer is known as the buffy coat.

For the purpose of producing buffy coat prepared platelets, buffy coatsare generally isolated and pooled by one of two methods depending on theformat of the bag in which the whole blood was collected. The firstmethod is known as the “top and bottom drain method” in which the baginto which the whole blood was collected has a top and bottom drain withone or more satellite containers attached to each end. An extractor(e.g., Optipress® Extractor from Fenwal) presses the bag flat such thatthe plasma layer is drained through the top drain and the red bloodcells are drained through the bottom drain. The extractor is designedsuch that the buffy coat containing primarily platelets and leukocyteswith a small volume of plasma and RBC, together comprising approximately30 to 60 mL of fluid volume, is retained within the bag. Approximately4-6 buffy coat units are pooled to make a therapeutic dose of plateletsfor transfusion to a patient. In pooling, individual buffy coat unitsare sterilely connected in a chain format often referred to as the“chain method” (e.g., the bottom drain of a bag is connected to the topdrain of the next bag, and so on.). A platelet protection solution orplasma can be sterilely connected to the chain and used to help rinseindividual buffy coat containers as the buffy coats are transferred tothe bottom pooling bag along with the platelet protection solution orplasma.

A second method for isolating and pooling buffy coat prepared plateletsutilizes a similar whole blood collection bag as used with PRP preparedplatelets. Following the isolation of the buffy coat in the whole bloodas described previously, the buffy coat is separated from the wholeblood by first removing the plasma into one of the attached satellitecontainers and transferring the buffy coat into a second attachedsatellite container, sometimes referred to as “milking the buffy coat”leaving the RBC in the original container. Approximately 4-6 buffy coatunits are pooled to make a therapeutic dose of platelets for transfusionto a patient. In pooling, individual buffy coat units are sterilelyconnected and pooled into a pooling container along with a plateletprotection solution or plasma. In this method, the pooling bag hasmultiple docks (e.g., like legs of a “spider”) to which the individualunits are connected. Each buffy coat unit is then transferred from theindividual bag into the pooling bag using the platelet protectionsolution or plasma as a rinsing agent to help reduce platelet loss inpooling. This pooling method is sometimes referred to as the “spidermethod” and can also be used with buffy coats prepared by top and bottomseparation.

Regardless of the method used to pool the individual units, the pooledbag undergoes centrifugation again. This centrifugation is a long, softspin in which a fraction containing platelets and the plasma/plateletprotection solution is formed at the top of the pooling bag and theremaining red blood cells and leukocytes become part of the bottomfraction. Using a plasma expresser or extractor, the top layer ofplatelets and plasma/platelet protection solution is transferred toanother bag resulting in a therapeutic dose of platelets.

Single donor platelets are platelets obtained from one donor by means ofcentrifugal separation in an automated apheresis machine in a quantitysufficient to constitute one or more therapeutic dose(s) for subsequenttransfusion to a patient(s). Platelets isolated by this method aregenerally known as single donor platelets because a therapeutic dose canbe collected from a single donor. In such a procedure, the donor's bloodflows from a point of venipuncture through a sterile centrifuge in whichthe platelets and a certain volume of plasma are centrifugally separatedand isolated, with the balance of the donor's blood being returned tothe donor through the initial venipuncture or a second point ofvenipuncture. Anticoagulant compositions, described herein, can be addedto the platelets or be present in the bag into which the platelets arecollected. Various automated apheresis devices are commerciallyavailable from companies such as Haemonetics Corporation (Braintree,Mass.), Terumo BCT (Lakewood, Colo.), Fenwal, Inc., Lake Zurich, Ill.,and Fresenius Kabi, Friedberg, Germany.

The collection of platelets by apheresis generally produces 2 plateletunits, wherein each unit contains approximately 200 to 300 mL of plasmaand approximately 3.5×10¹¹ platelets. Single donor platelets can bestored at 20-24° C. for about 5 days.

Apheresis collection kits often include two platelet collection bagssince most apheresis machines collect two units of platelets. Thecomposition of the present invention, as described herein, can beincluded in the platelet collection bags for apheresis machines or canbe added to the bag before, during or after collection of the plateletsusing a sterile connection technique. Platelet collection bags can bemanufactured with the composition of the present invention and furtherinclude additional components such as anticoagulant compositions asdescribed herein or known in the art.

After platelets are collected by apheresis, they can be suspended in thePPS of the present invention, as described herein.

The compositions and methods present invention can be used withplatelets isolated by any technique known in the art or developed in thefuture so long as a therapeutic concentration of platelets is obtained.

The present invention includes bags or containers including theβ-galactosidase inhibitor with or without the sialidase inhibitor and/orglycan-modifying composition, or the “inventive composition” asdescribed herein. Based on the platelet isolation process, the inventivecomposition can be included or manufactured with various plateletcollection bags. Platelet collection bags can be gas permeable or madefrom a plastic material such as PVC material. Platelet collections bagscan be used in the random donor collection process or in the singledonor collection process. With respect to the random donor collectionprocess, the inventive composition can be placed into the collection bagin which the platelet units are pooled; therefore the present inventionincludes a pooled collection bag having the inventive composition.

Similarly, in the single donor collection process, the inventivecomposition can be included in apheresis platelet collection bags. Alongwith the inventive composition, such bags include other components usedin the apheresis process such as anticoagulant compositions.

Conventional platelet bags or packs are formed of materials that aredesigned and constructed of a sufficiently permeable material tomaximize gas transport into and out of the pack (O₂ in and CO₂ out). Thepresent invention allows for storage of platelets at temperatures belowroom temperature or at room temperature, as further described herein.The methods described herein reduce or diminish the amount of CO₂generated by the platelets during storage. Accordingly, in anembodiment, the present invention further provides platelet containersthat are substantially non-permeable to CO₂ and/or O₂, which containersare useful particularly for cold storage of platelets. In anotherembodiment, the containers or bags include gas permeable containers.

With either collection process described above, the inventivecompositions can alternatively be added to the isolated platelets usinga sterile technique or connection. In such case, the inventivecomposition can be sold separately in a separate bag, container,syringe, tube or other similar blood collection medium.

In one embodiment, the composition of the present invention having theβ-galactosidase inhibitor with or without sialidase inhibitor and/orglycan-modifying agent, as further described herein, is contacted withthe platelets in a closed system, e.g., a sterile, sealed platelet packso as to avoid microbial contamination. Typically, a venipunctureconduit is the only opening in the pack during platelet procurement ortransfusion. Accordingly, to maintain a closed system during treatmentof the platelets with the composition of the present invention, suchcomposition is placed in a relatively small, sterile container which isattached to the platelet pack by a sterile connection tube (see e.g.,U.S. Pat. No. 4,412,835, the contents of which are incorporated hereinby reference). The connection tube may be reversibly sealed, or have abreakable seal, as will be known to those of skill in the art. After theplatelets are isolated, the seal to the container including thecomposition of the present invention is opened and the composition isintroduced into the platelet bag. In one embodiment, the composition ofthe present invention is contained in a separate container having aseparate resealable connection tube to permit the sequential addition ofthe composition to the platelets.

The Sialidase Inhibitor and the β-Galactosidase Inhibitor

Once the isolated platelets are obtained, platelets are treated with thecomposition of the present invention, which includes one or moreβ-galactosidase inhibitors, or both one or more β-galactosidaseinhibitors and one or more sialidase inhibitors; and optionally one ormore storage enhancing compositions such as glycan-modifying agents(e.g., monosaccharides such as arabinose, fructose, fucose, galactose,mannose, ribose, gluconic acid, galactosamine, glucosamine,N-acetylgalactosamine, muramic acid, sialic acid (N-acetylneuraminicacid), and nucleotide sugars such as cytidinemonophospho-N-acetylneuraminic acid (CMP-sialic acid), uridinediphosphate galactose (UDP-galactose) and UDP-galactose precursors suchas UDP-glucose). In some preferred embodiments, the glycan-modifyingagent is UDP-galactose and/or CMP-sialic acid. The composition of thepresent invention includes a “cocktail” in which more than one or acombination of these constituents is included. The phrase, “composition”or “inventive composition” refers to one or more β-galactosidaseinhibitors, or both one or more β-galactosidase inhibitors and one ormore sialidase inhibitors, and optionally one or more glycan-modifyingagents.

“Sialidase enzymes,” “sialidases,” also called “neuraminidases,” as usedherein, are glycoside hydrolase enzymes that cleave the glycosidiclinkages of neuraminic acids. Sialidase enzymes catalyze the hydrolysisof terminal sialic acid residues from platelet surface glycans. See FIG.8. Thus, sialidase inhibitors are used in several aspects of the presentinvention. Sialidase inhibitors reduce sialidase enzyme activity,prevent the hydrolysis of terminal sialic acid residues from plateletsurface glycans, preserve the integrity of platelet surface glycans,and/or maintain the function of platelets that are stored prior totransfusion.

“β-Galactosidase enzymes” as used herein, are glycoside hydrolaseenzymes that cleave the glycosidic linkages between sialic acid andβ-galactose. β-galactosidase enzymes catalyze the hydrolysis ofβ-galactose residues from platelet surface glycans. Thus,β-galactosidase enzymes inhibitors are used in several aspects of thepresent invention. β-galactosidase inhibitors reduce β-galactosidaseenzyme activity, prevent the hydrolysis of β-galactose residues fromplatelet surface glycans, assists in preserving the integrity ofplatelet surface glycans, and/or maintain the function of platelets thatare stored prior to transfusion.

Sialidase/neuraminidase enzymes are a large family, found in a range oforganisms. Neuraminidase enzymes are glycoside hydrolase enzymes (EC3.2.1.18) that cleave the glycosidic linkages of neuraminic acids. Acommonly known neuraminidase is a viral neuraminidase, a drug target forthe prevention of influenza infection. Other homologs are found inmammalian cells, and at least four mammalian sialidase homologs havebeen described in the human genome [e.g., Neu1 (Uniprot accessionnumbers: Q5JQI0, Q99519), Neu2 (Q9Y3R4), Neu3 (Q9UQ49.1), and Neu4(A8K056, B3KR54, Q8WWR8).

β-Galactosidase enzymes catalyze the hydrolysis of 3-galactosides intomonosaccharides. Substrates of different β-galactosidases includeβ-galactose, ganglioside GM1, lactosylceramides, lactose, and variousglycoproteins. β-Galactosidase is generally an exoglycosidase whichhydrolyzes the β-glycosidic bond formed between a galactose and itsorganic moiety.

As used herein, “sialidase inhibitor,” “neuraminidase inhibitor,” or“β-galactosidase inhibitor” can be any compound, small molecule,peptide, protein, aptamer, ribozyme, RNAi, or antisense oligonucleotideand the like. As used herein, “inhibit” means to interfere with thebinding or activity of an enzyme. Inhibition can be partial or total,resulting in a reduction or modulation in the activity of the enzyme asdetected.

For example, a sialidase or neuraminidase inhibitor/β-galactosidaseinhibitor according to the invention can be a protein, such as anantibody (monoclonal, polyclonal, humanized, and the like), or a bindingfragment thereof, directed against a neuraminidase protein. An antibodyfragment can be a form of an antibody other than the full-length formand includes portions or components that exist within full-lengthantibodies, in addition to antibody fragments that have been engineered.Antibody fragments can include, but are not limited to, single chain Fv(scFv), diabodies, Fv, and (Fab′)₂, triabodies, Fc, Fab, CDR1, CDR2,CDR3, combinations of CDR's, variable regions, tetrabodies, bifunctionalhybrid antibodies, framework regions, constant regions, and the like(see, Maynard et al., (2000) Ann. Rev. Biomed. Eng. 2:339-76; Hudson(1998) Curr. Opin. Biotechnol. 9:395-402). Antibodies can be obtainedcommercially, custom generated, or synthesized against an antigen ofinterest according to methods established in the art (Janeway et al.,(2001) Immunobiology, 5th ed., Garland Publishing).

Additionally, a sialidase or neuraminidase inhibitor/β-galactosidaseinhibitor can be a non-antibody peptide or polypeptide that binds aneuraminidase/galactosidase (e.g., a bacterial neuraminidase orbacterial galactosidase). A peptide or polypeptide can be a portion of aprotein molecule of interest other than the full-length form, andincludes peptides that are smaller constituents that exist within thefull-length amino acid sequence of a protein molecule of interest. Thesepeptides can be obtained commercially or synthesized via liquid phase orsolid phase synthesis methods (Atherton et al., (1989) Solid PhasePeptide Synthesis: a Practical Approach. IRL Press, Oxford, England).The peptide or protein-related sialidase or neuraminidaseinhibitors/β-galactosidase inhibitors can be isolated from a naturalsource, genetically engineered or chemically prepared. The type andsource of the β-galactosidase inhibitor, in embodiments that also have asialidase inhibitor, can be same, similar, or different from those ofthe sialidase inhibitor. These methods are well known in the art.

A sialidase or neuraminidase inhibitor/β-galactosidase inhibitor canalso be a small molecule that binds to a neuraminidase and disrupts itsfunction. Small molecules are a diverse group of synthetic and naturalsubstances generally having low molecular weights. They are isolatedfrom natural sources (for example, plants, fungi, microbes and thelike), are obtained commercially and/or available as libraries orcollections, or synthesized. Candidate sialidase or neuraminidaseinhibitor/β-galactosidase inhibitor small molecules can be identifiedvia in silico screening, fragment based drug discovery (FBDD), orhigh-through-put (HTP) screening of combinatorial libraries. Mostconventional pharmaceuticals, such as aspirin, penicillin, and manychemotherapeutics, are small molecules, can be obtained commercially,can be chemically synthesized, or can be obtained from random orcombinatorial libraries as described below (Werner et al., (2006) BriefFunct. Genomic Proteomic 5(1):32-6). In a preferred embodiment of theinvention, a small-molecule sialidase/neuramindase inhibitor is thesodium salt of 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA).

According to the present invention, the sialidase/neuraminidaseinhibitor can also be an FDA approved viral sialidase/neuraminidaseinhibitor, such as the viral sialidase/neuraminidase inhibitoroseltamivir also known asethyl(3R,4R,5S)-5-amino-4-acetamido-3-(pentan-3-yloxy)-cyclohex-1-ene-1-carboxylate(Tamiflu, Genentech, Cambridge, Mass.), zanamivir also known as((2R,3R,4S)-4-guanidino-3-(prop-1-en-2-ylamino)-2-((1R,2R)-1,2,3-trihydroxypropyl)-3,4-dihydro-2H-pyran-6-carboxylicacid) (Relenza; Glaxo Smith Kline, Research Triangle Park, N.C.); andPeramivir((1S,2S,3S,4R)-3-[(1S)-1-acetamido-2-ethyl-butyl]-4-(diaminomethylideneamino)-2-hydroxy-cyclopentane-1-carboxylicacid) (BioCryst, Birmingham, Ala.), or a variant thereof. For example,the viral sialidase/neuraminidase inhibitor, oseltamivir is an ethylester prodrug that can be purchased from Roche Laboratories (Nutley,N.J.). Amino acid sequences of FDA approved viralsialidase/neuraminidase inhibitors may also be derivatized, for example,bearing modifications other than insertion, deletion, or substitution ofamino acid residues, thus resulting in a variation of the originalproduct (a variant). These modifications can be covalent in nature, andinclude for example, chemical bonding with lipids, other organicmoieties, inorganic moieties, and polymers. For reviews on viralsialidase/neuraminidase inhibitors, see “The war against influenza:discovery and development of sialidase inhibitors.” Nature Reviews: DrugDiscovery (2007) 6 (12): 967-74. Klumpp et al., (2006) Curr. Top. Med.Chem. 6(5):423-34; Zhang et al., (2006) Mini Rev. Med. Chem.6(4):429-48; Jefferson et al., (2006) Lancet 367(9507):303-13; Alymovaet al., (2005) Curr Drug Targets Infect. Disord. 5(4):401-9; Moscona(2005) N. Engl. J. Med. 353(13):1363-73; De Clercq (2004) J. Clin.Virol. 30(2):115-33; Stiver (2003) CMAJ 168(1):49-56; Oxford et al.,(2003) Expert Rev. Anti. Infect. Ther. 1(2):337-42; Cheer et al., (2002)Am. J. Respir. Med. 1(2):147-52; Sidewell et al., (2002) Expert Opin.Investig. Drugs. 11(6):859-69; Doucette et al., (2001) Expert Opin.Pharmacother. 2(10):1671-83; Young et al., (2001) Philos. Trans. R. Soc.Lond. B. Biol. Sci. 356(1416):1905-13; Lew et al., (2000) Curr. Med.Chem. 7(6):663-72); Taylor et al., (1996) Curr. Opin. Struct. Biol. 19966(6): 830-7 and published U.S. Patent Application. Nos. 2009/0175805,2006/0057658, 2008/0199845 and 2004/0062801, the entirety of each ofwhich is incorporated herein by reference.

Accordingly, a “sialidase inhibitor” includes, but is not limited to oneor more of the following: fetuin; 2,3-dehydro-2-deoxy-N-acetylneuraminicacid (DANA); Oseltamivir(ethyl(3R,4R,5S)-5-amino-4-acetamido-3-(pentan-3-yloxy)-cyclohex-1-ene-1-carboxylate);Zanamivir((2R,3R,4S)-4-guanidino-3-(prop-1-en-2-ylamino)-2-((1R,2R)-1,2,3-trihydroxypropyl)-3,4-dihydro-2H-pyran-6-carboxylicacid); Laninamivir((4S,5R,6R)-5-acetamido-4-carbamimidamido-6-[(1R,2R)-3-hydroxy-2-methoxypropyl]-5,6-dihydro-4H-pyran-2-carboxylicacid); Peramivir((1S,2S,3S,4R)-3-[(1S)-1-acetamido-2-ethyl-butyl]-4-(diaminomethylideneamino)-2-hydroxy-cyclopentane-1-carboxylicacid); or a pharmaceutically acceptable salt thereof. Pharmaceuticallyacceptable salts of any of the foregoing can be used. In a still furtherpreferred embodiment, the sialidase inhibitor is the sodium salt of2,3-dehydro-2-deoxy-N-acetylneuraminic acid or a combination thereof.Sialidase inhibitors used with the present invention include those knownin the art or those later developed.

Accordingly, a “β-galactosidase inhibitor” includes, but is not limitedto one or more of the following: 1-deoxygalactonojirimycin (DGJ);N-(n-butyl)deoxygalactonojirimycin; N-(n-nonyl)deoxygalactonojirimycin;5-deoxy-L-arabinose; galactostatin bisulfate;3′,4′,7-trihydroxyisoflavone; D-ribonolactone;N-octyl-4-epi-β-valienamine; phenylethyl B-D-thiogalactopyranoside;difluorotetrahydropyridothiazinone; and 4-aminobenzyl1-thio-β-D-galactopryranoside; or a pharmaceutically acceptable saltthereof. In a still further preferred embodiment, the β-galactosidaseinhibitor is the 1-deoxygalactonojirimycin (DGJ). β-galactosidaseinhibitors used with the present invention include those known in theart or those later developed.

As used herein, a “glycan” or “glycan residue” is a polysaccharidemoiety on the surface of the platelet, exemplified by the GPIbαpolysaccharide. A “terminal” glycan residue is the monosaccharide/sugarresidue at the terminus of the polysaccharide chain, which typically isattached to polypeptides on the platelet surface. A glycan-modifyingagent includes an agent that modifies glycan residues on the platelet.The glycan-modifying agent repairs cleavage that occurs on the glycanresidue. In an embodiment, the glycan-modifying agent alters the sugarresidues of the polysaccharide chain of GPIbα on the surface of theplatelet.

Whereas β-galactosidase inhibitors, and sialidase inhibitors included insome of the embodiments, serve to preserve the integrity of the glycanstructures, and specifically the glycan termini, the glycan-modifyingagents serve to modify or repair glycans by the addition ofmonosaccharide(s) to the glycan. Thus, sialidaseinhibitors/β-galactosidase inhibitors and glycan-modifying agents servedistinct and complementary functions.

“Glycan-modifying agents,” as described herein, include monosaccharidessuch as arabinose, fructose, fucose, galactose, mannose, ribose,gluconic acid, galactosamine, glucosamine, N-acetylgalactosamine,muramic acid, sialic acid (N-acetylneuraminic acid), and nucleotidesugars such as cytidine monophospho-N-acetylneuraminic acid (CMP-sialicacid), uridine diphosphate galactose (UDP-galactose), and UDP-galactoseprecursors such as UDP-glucose. Glycan-modifying agents includeprecursors of CMP-sialic acid or UDP-galactose. In some preferredembodiments, the glycan-modifying agent is UDP-galactose or CMP-sialicacid, or both.

UDP-galactose is an intermediate in galactose metabolism, formed by theenzyme UDP-glucose-α-D-galactose-1-phosphate uridylyltransferase whichcatalyzes the release of glucose-1-phosphate from UDP-glucose inexchange for galactose-1-phosphate to make UDP-galactose. UDP-galactoseand sialic acid are available from several commercial suppliers such asSigma. In addition, methods for synthesis and production ofUDP-galactose are known in the art and described in the literature (seefor example, Liu et al., ChemBioChem 3, 348-355, 2002; Heidlas et al.,J. Org. Chem. 57, 152-157; Butler et al., Nat. Biotechnol. 8, 281-284,2000; Koizumi et al., Carbohydr. Res. 316, 179-183, 1999; Endo et al.,Appl. Microbiol., Biotechnol. 53, 257-261, 2000). UDP-galactoseprecursors are molecules, compounds, or intermediate compounds that maybe converted (e.g., enzymatically or biochemically) to UDP-galactose.One non-limiting example of a UDP-galactose precursor is UDP-glucose. Incertain embodiments, an enzyme that converts a UDP-galactose precursorto UDP-galactose is added to a reaction mixture (e.g., in a plateletcontainer).

In certain embodiments, the glycan-modifying agent is CMP-sialic acid ora CMP-sialic acid precursor. In further embodiments, the plateletcompositions comprising a CMP-sialic acid precursor further comprise anenzyme that converts the CMP-sialic acid precursor to CMP-sialic acid.In certain embodiments, the glycan-modifying agent is CMP-sialic acid.In certain embodiments, the glycan-modifying agent is UDP-galactose. Incertain embodiments, the glycan-modifying agents are CMP-sialic acid andUDP-galactose.

In certain embodiments, the sialidase inhibitor or the β-galactosidaseinhibitor is a protein. In further embodiments, the sialidaseinhibitor/β-galactosidase inhibitor is an antibody directed against aneuraminidase or β-galactosidase protein wherein the antibody ismonoclonal, polyclonal, humanized, or a binding fragment thereof. Incertain embodiments, the methods comprising a sialidaseinhibitor/β-galactosidase inhibitor that is a protein or an antibodyfurther comprise an effective amount of at least one glycan-modifyingagent. As mentioned, the nature, source, and other properties of theβ-galactosidase can be the same, similar, or different from those of thesialidase inhibitor, for embodiments in which a sialidase inhibitor isincluded. In certain embodiments, the glycan-modifying agent isCMP-sialic acid or a CMP-sialic acid precursor. In certain embodiments,the CMP-sialic acid precursor further comprises an enzyme that convertsthe CMP-sialic acid precursor to CMP-sialic acid. In certainembodiments, the glycan-modifying agent is UDP-galactose. In certainembodiments, the glycan-modifying agents are CMP-sialic acid andUDP-galactose.

Treating Platelets

The isolated platelets are treated by the composition of the presentinvention. Briefly, the overall process is described as follows. Withina time period of being isolated, the composition of the presentinvention is contacted with the isolated platelets to thereby obtain atreated platelet composition (e.g., referred to herein as a “plateletcomposition”). The platelet composition can be stored either at roomtemperature or in cold temperature and then warmed. The plateletcomposition is transfused into an individual in need of platelets and,as a result of the treatment with the inventive compositions, thetransfused platelets exhibit reduced bacterial proliferation and in vivoremain in circulation longer, and maintain hemostasis longer, ascompared to untreated platelets.

In an embodiment, the platelet composition includes one or moreβ-galactosidase inhibitors, or both one or more β-galactosidaseinhibitors and one or more of the sialidase inhibitors, as describedherein. In a certain embodiment, DANA is used as the sialidaseinhibitor. In an embodiment in which a cocktail of the composition ofthe present invention is used, in addition to one or moreβ-galactosidase inhibitors with or without one or more sialidaseinhibitors, one or more of the glycan-modifying agents, such asUDP-galactose and/or CMP-sialic acid, can be added.

After isolation of the platelets, as described herein or using othermethods known in the art, the platelets are treated with the compositionof the present invention. The composition of the present invention iscontacted with the isolated platelets in an amount that reducesβ-galactosidase activity, or both β-galactosidase activity and sialidaseactivity, inhibits bacterial proliferation, allows platelets to maintainhemostasis, and/or allows platelets to retain the ability to activateand form a clot. In an embodiment, an effective amount of either one ormore β-galactosidase inhibitors or one or more β-galactosidaseinhibitors in combination with one or more sialidase inhibitors and/oror one or more glycan-modifying agents is that amount that preserves oralters a sufficient number of glycan residues on the surface ofplatelets, such that when introduced to a population of platelets,reduces β-galactosidase activity or reduces both β-galactosidaseactivity and sialidase activity, inhibits bacterial proliferation,and/or increases circulation time of platelets or reduces the clearanceof the population of platelets in a mammal following transfusion of theplatelets into the mammal.

For example, an “effective amount” of either a sialidase inhibitor, aβ-galactosidase inhibitor, and/or a glycan-modifying agent to contactwith isolated platelets ranges from about 1 micromolar to about 10 mMfor each component, and most preferably about 200 micromolar to about3.0 mM (e.g., between about 1 and 10 micromolar, about 10 micromolar andabout 100 micromolar, about 100 and about 500 micromolar, about 500micromolar and about 1.0 mM, about 1.0 and about 1.5 mM, and about 1.5and about 2.5 mM). In another aspect, the concentrations are in a rangebetween about 10 micromolar to about 1000 micromolar, between about 100micromolar to about 150 micromolar, or between about 200 micromolar toabout 1200 micromolar.

When using the cocktail of the present invention, modification ofplatelets with a β-galactosidase inhibitor, a sialidaseinhibitor/β-galactosidase inhibitor, or a sialidaseinhibitor/β-galactosidase inhibitor in combination with one or moreglycan-modifying agents can be performed as follows. The population ofplatelets is contacted with the selected β-galactosidase inhibitor(s) orsialidase inhibitor(s) in combination with one or more β-galactosidaseinhibitor(s), and/or in combination with one or more glycan-modifyingagents. Multiple sialidase inhibitors, β-galactosidase inhibitors,and/or glycan-modifying agents (e.g., two, three, four or more) can beused simultaneously or sequentially. If used sequentially in time, thesialidase inhibitors, β-galactosidase inhibitors, and/orglycan-modifying agents are provided close enough in time to confer thedesired effect. In some embodiments, 0.1-500 mU/mL galactose transferaseor sialyl transferase is added to the population of platelets. Galactosetransfer can be monitored functionally using lectins such as FITC-ECL orsWGA binding. The goal of the glycan modification reaction is to reducesWGA binding to resting room temperature sWGA binding-levels. Galactosetransfer can be quantified using ¹⁴C-UDP-galactose. UDP-galactose ismixed with ¹⁴C-UDP-galactose to obtain appropriate galactose transfer.Platelets are extensively washed, and the incorporated radioactivitymeasured using a y-counter. The measured cpm (counts per minute) permitscalculation of the incorporated galactose. Similar lectin-bindingtechniques are applicable to monitoring sialic acid transfer.

The isolated platelets can be treated with the platelet composition in atime period before significant reduction in quality and/or hydrolysis ofsialic acid and/or β-galactose occurs. The addition of the compositionto the platelets can occur during the isolation process, shortly afterthe isolation process or within another time period.

As single donor platelets are removed from the donor's circulation byapheresis, as described herein, the composition of the present inventioncan be added in a sterile manner. For example, after the blood iscentrifuged by the apheresis machine and the platelets are separatedfrom the rest of the blood components, the composition of the presentinvention can be added into the bag containing platelets. In anotherembodiment, the collection bag into which the platelets are depositedafter centrifugation can already contain the composition of the presentinvention. In another embodiment, the composition of the presentinvention can be added to the bag into which the platelets are beingcollected simultaneously with the collection of the platelets. Once theplatelets come into contact with the composition of the presentinvention, the components can be mixed or agitated (e.g., bag turnedupside down and right side up) to ensure that the platelets come intocontact with inventive composition. In this example, little or no timepasses between the collection of the platelets and their treatment withthe inventive composition. Accordingly, contact of the inventivecomposition and the isolated platelets can occur during plateletdonation or soon after platelet isolation (e.g., between 1 minute andabout 120 minutes within platelet isolation).

In an embodiment, the composition of the present invention can be addedto the isolated platelets “immediately” after donation, within a certaintime period after donation, or “simultaneously” during donation. In anembodiment, the composition of the present invention is contacted withthe platelets in a range between about 1 minute and about 48 hours(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60 min, 1½ h, 2 h, 2½h, 3 h, 3½h, 4 h, 4½h, 5 h,5½h, 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h, or 48 h).

When random donor platelets are isolated from multiple donors, theinventive composition can be added after the platelets are isolated fromthe whole blood. In an embodiment, the addition of the inventivecomposition to the platelets can occur when the platelets from thedonors are pooled. The pooling bag that generally holds about 6 units ofrandom donor platelets can include the inventive composition so thatwhen the platelets are added to the pooling bag, the isolated plateletscome into contact with the composition. Alternatively, the compositioncan be sterilely connected to and introduced into the pooling bag duringor after the platelets are pooled. In an embodiment, the methods of thepresent invention include contacting the isolated platelets within 1hour to about 8 hours (e.g., between 1 and about 3 hours). In anembodiment, contacting the inventive composition with the isolatedplatelets should occur before platelets are refrigerated.

According to still yet another aspect of the invention, a device forcollecting and processing platelets is provided. The device has acontainer or bag for collecting platelets, wherein the container or bagincludes the composition of the present invention. In anotherembodiment, the device includes a container or bag that contains theisolated platelets and at least one satellite container or bag, whereinthe satellite container includes the composition of the presentinvention. The bag containing the platelets and the bag containing thecomposition of the present invention can be in sterile communicationwith one another.

The platelets, after being contacted with the inventive composition, canbe stored at room temperature or be refrigerated. In certain aspects,platelets are refrigerated to enable storage for longer periods of time.However, as further described herein, β-galactosidase inhibitors, aswell as the combination of β-galactosidase inhibitors with sialidaseinhibitors inhibit bacterial proliferation and allow platelets to bestored at room temperature.

In certain embodiments, the platelet compositions of the presentinvention include an effective amount of a β-galactosidase inhibitor, orβ-galactosidase inhibitor together with sialidase inhibitor, that isadded to a population of platelets after the platelets have beenobtained from a donor. In another embodiment, the novel plateletcomposition comprises an effective amount of a β-galactosidase inhibitoror β-galactosidase inhibitor together with sialidase inhibitor, that isadded to a population of platelets after the platelets have beenobtained from a donor and the resulting platelet composition is storedfor a period of time at room temperature without a substantial loss ofin vivo hemostatic activity and inhibition of bacterial proliferation.In another preferred embodiment, the novel platelet compositioncomprises an effective amount of β-galactosidase inhibitor or aβ-galactosidase inhibitor together with a sialidase inhibitor, that isadded to a population of platelets after the platelets have beenobtained from a donor; the resulting platelet composition is cooled to atemperature below room temperature; stored for a period of time at atemperature below room temperature and rewarmed back to room temperaturewithout a substantial loss in vivo hemostatic activity.

The terms “cooling,” “cold temperature,” “temperature below roomtemperature,” and “temperature below ambient temperature,”interchangeably refer to any temperature between 28° C. and −100° C. Inany of the embodiments of the invention described herein, thetemperature is alternatively selected from the group of temperaturesconsisting of 27° C., 26° C., 25° C., 24° C., 23° C., 22° C., 21° C.,20° C., 19° C., 18° C., 17° C., 16° C., 15° C., 14° C., 13° C., 12° C.,11° C., 10° C., 9° C., 8° C., 7° C., 6° C., 5° C., 4° C., 3° C., 2° C.,1° C., 0° C., −1° C., −2° C., −3° C., −4° C., −5° C., −6° C., −7° C.,−8° C., −9° C., and −10° C. In some embodiments, the plateletpreparation is stored at a temperature of less than about 15° C.,preferably less than 10° C., and more preferably less than 5° C. In someother embodiments, the platelet preparation is stored at roomtemperature. In other embodiments, the platelets are frozen, e.g., 0°C., −20° C., or −80° C., or cooler.

As used in all of the aspects and embodiments of the invention herein,the term “period of time” refers to a duration of time during whichplatelets or platelet compositions are stored at any given temperature.The term “period of time” can range from seconds to minutes to hours todays to weeks. In preferred embodiments, the term “period of time”refers a number of hours including about 3 to about 120 hours, e.g., 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109,110, 111, 112, 113, 114, 115, 116, 117, 118, 119, and 120 hours. Incertain embodiments the period of time for which treated platelets canbe stored include about 1 and about 30 days (e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, and 30).

In an embodiment, treated platelets can be stored at room temperaturefor about 1 to about 14 days (e.g., about 7 days). In an aspect, theplatelets can be refrigerated on any day or days during storage.

In various other embodiments, the treated platelets are stored at roomtemperature. Treatment with one or more β-galactosidase inhibitors, or acombination of both one or more β-galactosidase inhibitors and one ormore sialidase inhibitors, and optionally for any of the aboveembodiments, one or more glycan-modifying agents preserves/modifies theplatelet population, i.e., preserves or improves the hemostatic functionof the platelet population following transfusion into a mammal, andreduces the incidence of storage lesions in room temperature storedplatelets, when compared to untreated platelet samples over a period oftime following treatment. Treated platelet samples stored at or belowroom temperature are thus suitable for autologous or heterologoustransfusion after extended periods of storage time, in an embodiment,for at least about 2 days, at least about 3 days, at least about 4 days,at least about 5 days, at least about 6 days, at least about 7 days, atleast about 8 days, at least about 9 days, at least about 10 days, atleast about 11 days, at least about 12 days, at least about 13 days, atleast about 14 days, at least about 21 days, or at least about 28 days.

As used in all of the aspects and embodiments of the invention herein,the term “warmed slowly” refers a gradual rate of warming (e.g., 0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or10° C. per hour or per day). As described herein, any of the aspects orembodiments of the invention further comprises a step of warming thetreated platelet preparation above room temperature, for example, bywarming the platelets to 37° C. Warming can occur gradually or bystepwise temperature increases. It is preferable to warm eitherroom-temperature-stored or cold-stored and treated platelet populationby slow addition of heat, and with continuous gentle agitation such asis common with the rewarming of blood products. A blood warming deviceis disclosed at WO/2004/098675 and is suitable for rewarming a treatedplatelet population from cold storage conditions.

Inhibition of Bacterial Proliferation and Pathogen-Induced PlateletDegradation

This invention provides a novel method to reduce pathogen-inducedplatelet degradation and inhibit pathogen growth/propagation byinhibiting β-galactosidases, or both β-galactosidases and sialidases,any of which may be from a pathogenic source. Sialidase and/orβ-galactosidase inhibitors exhibit anti-microbial properties thatprevent pathogenic proliferation.

The term “pathogen” as used herein, refers to one or more microorganismsor the like that cause infection as described in (Dodd, R. Y. New Engl.J. Med. 327:419-421 (1992); Soland, E. M., et al. J. Am. Med. Assoc.274:1368-1373 (1995) and Schreiber, G. B., et al. New Engl. J. Med.334:1685-1690 (1996)). Exemplary pathogens include, but are not limitedto a virus, bacteria, parasite, protozoa, or fungus. Examples of virusesinclude, but are not limited to Herpes simplex virus, HIV, hepatitis,hepatitis A, hepatitis B, hepatitis C, Respiratory syncycial virus, bluetongue virus, and bovine diarrhea virus. Virus also includesCytomegalovirus, Epstein-Barr virus, Herpes Simplex type I and IIviruses, and other viruses that circulate freely in the blood, as wellas cell-associated viruses. Fungus includes, but is not limited to e.g.,Aspergillus. And typical parasites include, but are not limited to, forexample: Ameoba, Plasmodiunm, Leishmania, Mycosus profundus,Trypanosoma, Spirochete, and Arbovius.

Bacteria commonly associated with platelets and whose proliferation isinhibited by a sialidase inhibitor and/or a β-galactosidase inhibitorinclude, but are not limited to Aspergillus, Bacillus sp, Bacteroideseggerthii, Candida albicans, Citrobacter sp, Clostridium perfringens,Corynebacterium sp, Diphtheroid, Enterobacter aerogenes, Enterobacteramnigenus, Enterobacter cloacae, Enterococcus avium, Enterococcusfaecalis, Escherichia coli, Fusobacterium spp., Granulicatella adiacens,Heliobacter pylori, Klebsiella sp, (K. pneumonia, K. oxytoca),Lactobacillus sp, Listeria sp, Micrococcus sp, Peptostreptococcus,Proteus vulgaris, Pseudomonas sp, Pseudomys oralis, Propionibacteriumsp, Salmonella sp, Serratia sp, Staplhylococcus sp (Coagulase-negativeStaphylococcus, Staphylococcus epidermidis, Staphylococcus aureus),Streptococcus sp, (S. gallolyticus, S. bovis, S. pyogenes, S. viridans),Serratia marcescens, and Yersinia enterocolitica.

The term “pathogen-induced platelet degradation” as used herein, refersto any degree of platelet degradation, decrease in hemostatic activity,or increase in the clearance rate of platelets that is caused by one ormore pathogens.

The term “detrimental effect” as used herein, can refer to a detrimentaleffect upon the viability of platelets (e.g., an increase in plateletdegradation, decrease in hemostatic activity, or increase in theclearance rate of platelets) that is caused by one or more pathogens.The term “detrimental effect” as used herein, can also refer to thedetrimental effect upon the patient (e.g., the consequences of theinfection itself) that is caused by one or more pathogens such assepsis.

The term “bacterial contamination” as used herein, refers tocontamination by any of the above-described bacterial pathogens or bynon-pathogenic bacteria that are capable of producing bacteria-derivedsialidase. “Inhibiting bacterial proliferation” refers to reducingand/or inhibiting the growth of bacteria in a platelet preparation.

The term “bacteria-derived sialidase” as used herein, refers tosialidase that is produced by bacteria. The inhibition of“bacteria-derived sialidase” as used herein, can optionally inhibitplatelet-derived sialidase and/or patient-derived sialidase in additionto the inhibition of bacteria-derived sialidase.

The invention, in other aspects, provides a novel method to inhibitionof bacterial proliferation in a platelet preparation by obtaining apopulation of platelets from a donor and contacting the platelets withan effective amount of the inventive compositions e.g., aβ-galactosidase inhibitor or β-galactosidase inhibitor together withsialidase inhibitor. In a preferred embodiment, the methods of thepresent invention further include storing the treated plateletcomposition for a period of time at room temperature without asubstantial loss of in vivo hemostatic activity. Alternatively, asdescribed herein, the treated platelets or the resulting plateletcompositions can be cooled to a temperature below room temperature;stored for a period of time at a temperature below room temperature, andrewarmed back to room temperature without a substantial loss of in vivohemostatic activity.

Preferred embodiments of the inventive method to reduce pathogen growthin a platelet preparation, as described herein, include contactingplatelets with an effective amount of a β-galactosidase inhibitor, or aβ-galactosidase inhibitor together with a sialidase inhibitor asdescribed herein, and optionally with an effective amount of at leastone glycan-modifying agent, as described herein.

The anti-proliferative inhibition of bacteria by the β-galactosidaseinhibitor with or without the sialidase inhibitor allows platelets to bestored for longer with a reduced risk of bacterial contamination, andfor the time period described herein.

Bacterial contamination of platelets is a concern because it causessepsis in patients receiving them. Bacterial contamination can be theresult of non-sterile techniques in obtaining blood and/or plateletsfrom the donor, or in poor handling of the platelets after donation.Despite good sterile techniques in obtaining donated blood or platelets,bacteria can still persist in the platelet preparation. For example,even though a technician uses an antibacterial agent to clean the skinat the site of donation, bacteria can be embedded within the layers ofthe skin, i.e., intradermally. So, upon penetration of the skin with aneedle, bacterial contamination of the platelet donation can occur. As aresult, bacterial testing at the point of care (e.g., at the time therecipient receives the platelets) is performed to reduce the risk ofsepsis.

Additionally, bacterial contamination can result in the formation ofbiofilm on the interior surfaces of blood containers/bags. The biofilmformation is the result of bacteria attaching to the interior surface ofthe bag and proliferating using the surface as a support. As thebacterial proliferation increases, the biofilm formation also increases.

Accordingly, contacting the platelet preparation with β-galactosidaseinhibitors or both β-galactosidase inhibitors and sialidase inhibitorsprovides unexpected anti-proliferative inhibition of bacteria and areduction in biofilm formation on the interior surface of the plateletbag. Using the methods described herein the platelet preparation iscontacted with an effective amount of one or more β-galactosidaseinhibitors with or without one or more sialidase inhibitors, whichinhibits endogenous platelet enzymes but also bacterial enzymes. Thistreatment of platelets results in prolonged storage of platelets withreduced bacterial growth/proliferation, which provides platelets with anincreased survival and hemostasis in vivo after transfusion into arecipient.

Encompassed in the method of the present invention is testing forbacterial proliferation at one or more time points to determine thatbacterial proliferation is in fact inhibited before being transfusedinto the recipient. Bacterial testing can occur at a single time point(e.g., at the point of care) and the results can be compared to astandard to determine if bacterial proliferation has occurred in thetreated platelets to be transferred. Additionally, bacterial testing ofthe treated platelets can occur at more than one time point to assess ifthe particular sample has exhibited inhibition of bacterialproliferation. An increase in bacterial proliferation or the presence ofbacterial proliferation indicates that the treated platelets arecontaminated and cannot be used for transfusion. The absence ofbacterial proliferation indicates that the treated platelets can be usedfor transfusion. Using the β-galactosidase inhibitor, or theβ-galactosidase inhibitor together with the sialidase inhibitor of thepresent invention results in treated platelets that are suitable fortransfusion.

A number of tests exist to determine the presence of bacterialcontamination in a treated platelet preparation. Bacteria can be testedby the presence of a polypeptide or protein that is common to bacteriaand not found in platelets, by culture techniques, Gram staining,scanning techniques, the presence of nucleic acid that is conserved inbacteria, scans, and the like.

A commonly used test in determining bacterial contamination of aplatelet preparation is the Pan Genera Detection (PDG® test) (VeraxBiomedical, Incorporated, Worcester Mass.). The PGD® test can detect anarray of bacteria in blood components. This broad detection is based onthe existence of shared, or conserved, antigens that are common to thecell walls of the two broad classes of bacteria: lipoteichoic Acids onGram-positive bacteria and lipopolysaccharides on Gram-negativebacteria. The test targets these conserved Gram-positive andGram-negative antigens to test biological samples for a broad range ofbacterial contaminants by using binding agents to directly bind to thesetargets. Although the level or presence of the specific bacteria is notdetermined by this test, the test does determine the presence of anumber of bacteria in the platelet preparation.

Culture methods can be employed to determine the presence or absence ofbacterial contamination and/or bacterial proliferation. One commerciallyavailable test is referred to as the BacT/ALERT® test (bioMérieux, Inc.,Durham, N.C.). Bacterial detection is based on the evolution of carbondioxide by proliferating bacteria. A carbon-dioxide-sensitive liquidemulsion sensor at the bottom of the culture bottle changes color and isdetected through alteration of light reflected on the sensor.BacT/ALERT® test detects the presence of a number of bacteria, fungi,and yeasts.

Another method for bacterial detection involves measuring the oxygencontent in a platelet preparation sample. An example is the Pall eBDStest (Pall Corporation, Port Washington, N.Y.). The approach todetection measures the oxygen content of air within the sample pouch asa surrogate marker for bacteria. An oxygen analyzer is used to measurethe percent of oxygen in the headspace gas of the pouch or bag havingthe platelets. If bacteria are present in the platelet sample collected,an increasing amount of oxygen is consumed through the metabolicactivity and proliferation of the bacteria in the sample duringincubation, resulting in a measurable decrease in oxygen content of theplasma as well as the air within the sample pouch.

A more conventional method for determining the presence of bacterialproliferation is a platelet preparation is Gram staining. Gram stainingallows one to differentiate bacterial species into classes(Gram-negative or Gram-positive) in an effort to begin to identify themicroorganism. The test detects peptidoglycan, a glycan in the cell wallof the bacteria.

A sample from the treated platelet preparation can be obtained andcultured to determine if any bacteria are present. The growth media isinoculated or plated with the sample and under controlled conditionssuitable for bacterial growth. Bacteria can be grown and identified.

Other methods known in the art or developed in the future can be used todetermine bacterial proliferation in the treated platelet preparation ofthe present invention.

The methods of the present invention involve reducing bacterialproliferation and/or biofilm formation by contacting the plateletpreparation with an effective amount of one or more β-galactosidaseinhibitors, or both one or more β-galactosidase inhibitors and one ormore sialidase inhibitors. The bacterial proliferation is reduced, ascompared to a standard or to another assessment taken at a differenttime point. The methods described herein reduce bacterial proliferationand/or biofilm formation by at least about 5% (e.g., by about 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%). In an embodiment, themethods of the present invention completely inhibit bacterialproliferation and/or biofilm formation, as compared to that at the timeof treatment of the platelet preparation with the β-galactosidaseinhibitor, or both the β-galactosidase inhibitor and the sialidaseinhibitor.

Storage of Platelets

The invention embraces a method for increasing the storage time ofplatelets. During storage with the β-galactosidase inhibitor, or withthe β-galactosidase inhibitor and the sialidase inhibitor describedherein, platelets can be stored with reduced β-galactosidase, or reducedsialidase/β-galactosidase activity, inhibited bacterial proliferation,and without substantial loss of platelet function or hemostatic activitysuch as the loss of the ability to circulate or without an increase inthe rate of platelet clearance.

The platelets are collected from blood by standard techniques known tothose of ordinary skill in the art, as described herein. The storagecomposition includes at least one β-galactosidase inhibitor, or at leastone of both a sialidase inhibitor and a β-galactosidase inhibitor; andoptionally, at least one glycan-modifying agent in an amount sufficientto reduce platelet clearance. In some embodiments, the storagecomposition further comprises an enzyme that catalyzes the modificationof a glycan moiety on the platelet.

The invention, in certain aspects, provides a novel method of storing aplatelet composition in which the steps includes obtaining a populationof platelets from a donor and treating the platelets with an effectiveamount of one or more β-galactosidase inhibitors or both one or moreβ-galactosidase inhibitors and one or more sialidase inhibitors; andoptionally one or more glycan modifying agents. In an embodiment, thenovel method of storing a platelet composition involves obtaining apopulation of platelets from a donor; adding an effective amount of aβ-galactosidase inhibitor or both an effective amount of aβ-galactosidase inhibitor and a sialidase inhibitor to the population ofplatelets and storing the resulting platelet composition for a period oftime at room temperature without a substantial loss in vivo hemostaticactivity. In another embodiment, the novel method of storing a plateletcomposition encompasses obtaining a population of platelets from adonor; adding an effective amount of a β-galactosidase inhibitor, orboth a β-galactosidase inhibitor and a sialidase inhibitor, to thepopulation of platelets; cooling the resulting platelet composition to atemperature below room temperature; storing the platelet composition fora period of time at a temperature below room temperature and rewarmingthe platelet composition back to room temperature without a substantialloss in vivo hemostatic activity. In further embodiments, the plateletcomposition is rewarmed slowly. In certain embodiments, the plateletcomposition retains substantially normal hemostatic activity whentransfused into a mammal after storage. In further embodiments, theplatelet composition when transfused into a mammal after storage, has acirculation half-life of about 5% or greater than the circulationhalf-life of untreated platelets. In certain preferred embodiments, theplatelet composition is suitable for transfusion into a human afterstorage.

In accordance with the invention, following treatment with aβ-galactosidase inhibitor or both a β-galactosidase inhibitor and asialidase inhibitor, the population of treated platelets can be storedat room temperature or chilled without the deleterious effects(cold-induced platelet activation) experienced upon chilling ofuntreated platelets. The preservation and/or selective modification ofglycan moieties reduce clearance, thus permitting longer-term storagethan is presently possible. In one aspect, one or more β-galactosidaseinhibitors or both one or more β-galactosidase inhibitors and one ormore sialidase inhibitors are added to the population of platelets thatare kept between about room temperature (between about 20° C. and 25°C.) and 37° C. As used herein, chilling refers to lowering thetemperature of the population of platelets to a temperature that is lessthan about 25° C. In some embodiments, the platelets are chilled to atemperature that is less than about 15° C. In some preferredembodiments, the platelets are chilled to a temperature ranging frombetween about 0° C. to about 4° C. Chilling also encompasses freezingthe platelet preparation, i.e., to temperatures less than 0° C., −20°C., −50° C., and −80° C. or cooler. Processes for the cryopreservationof cells are well known in the art.

In some embodiments, the population of platelets is stored at roomtemperature for at least 3 days. For example, the population of treatedplatelets is stored at room temperature for at least 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,and 28 days or longer.

Additionally, in certain aspects, a population of treated platelets canbe stored chilled for at least 3 days. A population of treated plateletsis stored chilled e.g., for at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 days orlonger.

Transfusion of Platelets into Mammals (e.g., Humans)

After storage, the present invention, in some aspects, provides a methodof transfusing a patient with a treated platelet composition having oneor more β-galactosidase inhibitors, or both one or more β-galactosidaseinhibitors and one or more sialidase inhibitors, wherein the plateletcomposition was prepared according to the methods described herein.Similarly, using these steps, the present invention provides a novelmethod for mediating hemostasis in a mammal.

Additionally, the present invention relates to methods for increasingthe circulation time of platelets, or reducing the clearance ofplatelets. The circulation time of a population of platelets is definedas the time when one-half of the platelets in that population are nolonger circulating in a mammal after transfusion into that mammal.

As used herein, “clearance” means removal of the treated platelets fromthe blood circulation of a mammal (such as but not limited to bymacrophage phagocytosis). More specifically, clearance of a populationof platelets refers to the removal of a population of platelets from aunit volume of blood or serum per unit of time. Reducing the clearanceof a population of platelets refers to preventing, delaying, or reducingthe clearance of the population of platelets or the rate at whichplatelets clear.

Patients in need of platelet transfusion include those with e.g.,anemia, thrombocytopenia, dysfunctional platelet disorders, activeplatelet-related bleeding disorders, or serious risk of bleeding (e.g.,prophylactic use). Patients with certain medical conditions at timesrequire platelet transfusion. Such conditions include, among others:leukemia, myelodysplasia, aplastic anemia, solid tumors, congenital oracquired platelet dysfunction, central nervous system trauma. Patientsundergoing extracorporeal membrane oxygenation or cardiopulmonary bypassalso receive platelet transfusions.

In one aspect of the invention, the method for increasing circulationtime of an isolated population of platelets involves contacting anisolated population of platelets with at least one β-galactosidaseinhibitor, or both at least one β-galactosidase inhibitor and at leastone sialidase inhibitor, in an amount effective to reduce the clearanceof the population of platelets. As used herein, a population ofplatelets refers to a sample having one or more platelets.

Reducing the clearance of a platelet encompasses reducing the clearanceof platelets that results after storage of the platelets at or belowroom temperature. Reducing the clearance of a platelet can result fromreducing storage lesions obtained at or below room temperature, orreducing “cold-induced platelet activation” that occurs upon the coldstorage of platelets. Cold-induced platelet activation is a term havinga particular meaning to one of ordinary skill in the art. Cold-inducedplatelet activation can be manifested by changes in platelet morphology,some of which are similar to the changes that result following plateletactivation. The structural changes indicative ofroom-temperature-induced or cold-induced platelet activation are mosteasily identified using techniques such as light or electron microscopy.On a molecular level, platelet activation results in actin bundleformation and a subsequent increase in the concentration ofintracellular calcium. Actin-bundle formation is detected using, forexample, electron microscopy. An increase in intracellular calciumconcentration is determined, for example, by employing fluorescentintracellular calcium chelators. Many of the above-described chelatorsfor inhibiting actin filament severing are also useful for determiningthe concentration of intracellular calcium (Tsien, R., 1980, supra.).Accordingly, various techniques are available to determine whether ornot platelets have experienced room-temperature-induced or cold-inducedactivation.

The addition of a β-galactosidase inhibitor or sialidase inhibitor andβ-galactosidase inhibitor to platelets prevents the hydrolysis ofβ-galactose or sialic acid/β-galactose residues, respectively, from thetermini of glycans and preserves the structures of glycan moieties onplatelets, resulting in diminished clearance of treated platelets. Thiseffect can be measured, for example, using either an in vitro systememploying differentiated THP-1 cells or mouse macrophages, isolated fromthe peritoneal cavity after thioglycolate injection stimulation. Therate of clearance of treated platelets compared to untreated plateletscan be determined. To test clearance rates, the treated platelets arefed to the macrophages and ingestion of the platelets by the macrophagesis monitored. Reduced ingestion of treated platelets as compared tountreated platelets (1.2-fold or greater) indicates successfulmodification of the glycan moiety for the purposes described herein.

Also, the addition of a β-galactosidase inhibitor or both aβ-galactosidase inhibitor and a sialidase inhibitor to plateletsinhibits bacterial proliferation, which in turn, reduces plateletclearance and prevents sepsis. Assessment of bacterial proliferation isdescribed herein.

In certain embodiments of the invention, the circulation time of thepopulation of platelets is increased by at least about 10%, 20%, 25%,30%, or 40%. In some embodiments, the circulation time of the populationof platelets is increased by at least about 50% to about 100%. In stillyet other embodiments, the circulation time of the population ofplatelets is increased by about 150% or greater.

Platelet Compositions

After being subjected to the β-galactosidase inhibitor, or to theβ-galactosidase inhibitor and the sialidase inhibitor as describedherein, the platelets are treated and are referred to herein as“platelet compositions” or “treated platelets.” The present inventionincludes a novel platelet composition comprising one or moreβ-galactosidase inhibitors or both one or more β-galactosidaseinhibitors and one or more sialidase inhibitors, as described herein. Inanother embodiment, the novel platelet composition further comprises aneffective amount of at least one glycan-modifying agent. The treatedplatelets have a plurality of intact glycan molecules on the surface ofthe platelet that would otherwise have been cleaved withoutβ-galactosidase inhibitor treatment or without β-galactosidase inhibitorand sialidase inhibitor treatment. The glycan molecules of the plateletcomposition of the present invention include those in which sialicacid/β-galactose cleavage is prevented and the glycan molecules remainintact. In the event that sialic acid/β-galactose is cleaved, then theglycan-modifying agents (e.g., CMP-sialic acid, or UDP-galactose, orboth) allow for sialic acid/β-galactose additions to the terminal sugarresidues, or galactosylation of the terminal sugar residues, or bothsialylation and galactosylation of the terminal sugar residues. In someembodiments, the modified glycan moieties are GPIbα molecules. Theinvention also encompasses a platelet composition in a storage medium.In some embodiments, the storage medium can be a pharmaceuticallyacceptable carrier.

In some embodiments, the terminal glycan molecules so modified are GPIbαmolecules. The treated platelets include glycan structures with terminalGPIbα molecules that following treatment have terminal galactose orsialic acid attached to the GPbα molecules. In another aspect, theinvention provides a platelet composition comprising a plurality oftreated platelets. In some embodiments, the platelet composition furthercomprises a storage medium. In some embodiments, the plateletcomposition further comprises a pharmaceutically acceptable carrier.

In some embodiments, the population of platelets treated according tothe inventive methods described herein demonstrates inhibited bacterialproliferation and substantially normal hemostatic activity, preferablyafter transfusion into a mammal. In some embodiments, the population ofplatelets treated according to the inventive methods described hereindemonstrates reduced bacterial proliferation and improved hemostaticactivity, relative to a similarly stored but untreated population ofplatelets.

In a further preferred embodiment, the novel platelet composition, asdescribed above, provides a stable platelet preparation. In certainembodiments, the stable platelet preparation of the invention is capableof being stored for at least 24-360 hours, and the platelet preparationis suitable for administration/transfusion to a human after storagewithout significant loss of hemostatic function or without a significantincrease in platelet clearance in the human as compared to the same foruntreated platelets. In certain preferred embodiments, the stableplatelet preparation is capable of being cold-stored. In certain otherpreferred embodiments, the platelets are capable of being stored at roomtemperature without substantial reduction in biological activitycompared to the same for non-treated platelets.

The invention, in other aspects, provides compositions comprising anovel platelet composition, as described herein, and further comprisingat least one pharmaceutically acceptable excipient. A “pharmaceuticallyacceptable excipient,” as used herein, includes any and all solvents,diluents, or other liquid vehicle, dispersion or suspension aids,surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives, antioxidants, solid binders, lubricants, and thelike, as suited to the particular dosage form desired. Remington'sPharmaceutical Sciences, Sixteenth Edition, E. W. Martin (MackPublishing Co., Easton, Pa., 1980) discloses various excipients used informulating pharmaceutical compositions and known techniques for thepreparation thereof. Except insofar as any conventional excipient mediumis incompatible with the compounds of the invention, such as byproducing any undesirable biological effect or otherwise interacting ina deleterious manner with any other component(s) of the pharmaceuticalcomposition, its use is contemplated to be within the scope of thisinvention.

In certain embodiments, the platelet composition is suitable fortransfusion into a human patient afflicted with a bleeding disorder oranemia. In preferred embodiments, the platelet composition can be storedfor at least 5 days with inhibited bacterial proliferation prior toadministration to a human, and wherein the composition can be transfusedinto a human after storage without significant loss of hemostaticfunction or without a significant increase in platelet clearance in thehuman as compared to untreated platelets.

The term “pharmaceutically acceptable” means a non-toxic material thatdoes not interfere with the effectiveness of the biological activity ofthe platelets and that is a non-toxic material that is compatible with abiological system such as a cell, cell culture, tissue, or organism.Pharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers, and other materials which are wellknown in the art, for example, a buffer that stabilizes the plateletpreparation to a pH of 7.3-7.4, the physiological pH of blood, is apharmaceutically acceptable composition suitable for use with thepresent invention.

The invention further embraces a method for making a pharmaceuticalcomposition for administration to a mammal. In a preferred embodiment,the novel pharmaceutical composition comprising platelets furthercomprises an effective amount of a β-galactosidase inhibitor or both aβ-galactosidase inhibitor and a sialidase inhibitor that are added to apopulation of platelets after the platelets have been obtained from adonor and the resulting platelet composition is stored for a period oftime at room temperature without a substantial loss in vivo hemostaticactivity. In another preferred embodiment, the novel pharmaceuticalcomposition comprising platelets further comprises an effective amountof a β-galactosidase inhibitor or both a β-galactosidase inhibitor and asialidase inhibitor that are added to a population of platelets afterthe platelets have been obtained from a donor; the resulting plateletcomposition is cooled to a temperature below room temperature; storedfor a period of time at a temperature below room temperature andrewarmed back to room temperature without a substantial loss in vivohemostatic activity. In some embodiments, the method of preparing thenovel pharmaceutical compositions comprising platelets comprisesneutralizing, removing or diluting the β-galactosidase inhibitor and/orsialidase inhibitors/β-galactosidase inhibitor and/or glycan-modifyingagent(s) and/or the enzyme(s) that preserve and/or catalyze themodification of the glycan moiety, and placing the treated plateletpreparation in a pharmaceutically acceptable carrier. In one preferredembodiment, the platelets are stored at room temperature (about 22° C.)prior to and during neutralization or dilution. In another preferredembodiment, the platelets are chilled, stored and then warmed to roomtemperature (about 22° C.) prior to neutralization or dilution. In someembodiments, the platelets are contained in a pharmaceuticallyacceptable carrier prior to contact with the β-galactosidase inhibitorand/or sialidase inhibitors/β-galactosidase inhibitor and/orglycan-modifying agent(s) and/or the enzyme(s) that preserve and/orcatalyze the modification of the glycan moiety and it is not necessaryto place the platelet preparation in a pharmaceutically acceptablecarrier following neutralization or dilution.

As used herein, the terms “neutralize” or “neutralization” refer to aprocess by which β-galactosidase inhibitors, the β-galactosidaseinhibitors and the sialidase inhibitors, and/or glycan-modifyingagent(s) and/or the enzyme(s) that preserve and/or catalyze themodification of the glycan moiety are rendered substantially incapableof glycan modification of the glycan residues on the platelets, or theirconcentration in the platelet solution is lowered to levels that are notharmful to a mammal, for example, to less than 50 micromolar for theglycan-modifying agents. In some embodiments, the chilled platelets areneutralized by dilution, e.g., with a suspension of red blood cells.Alternatively, the treated platelets can be infused into the recipient,which is equivalent to dilution into a red blood cell suspension. Thismethod of neutralization advantageously maintains a closed system andminimizes damage to the platelets. In a preferred embodiment, noneutralization is required.

An alternative method to reduce toxicity is by inserting a filter in theinfusion line, the filter containing, e.g., activated charcoal or animmobilized antibody, to remove the β-galactosidase inhibitors, thesialidase inhibitors if present, and/or glycan-modifying agent(s) and/orthe enzyme(s) that preserve and/or catalyze the modification of theglycan moiety.

Either or all of the β-galactosidase inhibitors, sialidase inhibitors ifpresent, and/or glycan-modifying agent(s) and/or the enzyme(s) thatpreserve and/or catalyze the modification of the glycan moiety also maybe removed or substantially diluted by washing the treated platelets inaccordance with standard clinical cell washing techniques.

The invention further provides a method for mediating hemostasis in amammal. The method includes administering the above-described treatedplatelets. The transfusion of the treated platelets or pharmaceuticalcomposition can be done in accordance with standard methods known in theart. According to one embodiment, a human patient is transfused with redblood cells before, after, or during administration of the treatedplatelets. The red blood cell transfusion serves to dilute theadministered, treated platelets, thereby neutralizing theβ-galactosidase inhibitors, the sialidase inhibitors if present, and/orglycan-modifying agent(s) and/or the enzyme(s) that preserve and/orcatalyze the modification of the glycan moiety.

The dosage regimen for mediating hemostasis using the treated plateletsis selected in accordance with a variety of factors, including the type,age, weight, sex and medical condition of the subject, the severity ofthe disease, the route and frequency of administration. An ordinarilyskilled physician or clinician can readily determine and prescribe theeffective amount of treated platelets required to mediate hemostasis.

The dosage regimen can be determined, for example, by following theresponse to the treatment in terms clinical signs and laboratory tests.Examples of such clinical signs and laboratory tests are well known inthe art and are described, for example in, HARRISON'S PRINCIPLES OFINTERNAL MEDICINE, 15th Ed., Fauci A S et al., eds., McGraw-Hill, NewYork, 2001.

For example, to determine the optimal concentrations and conditions forpreventing room-temperature-induced activation or cold-inducedactivation of platelets by treating them with one or moreβ-galactosidase inhibitors or both one or more β-galactosidaseinhibitors and one or more sialidase inhibitors; and optionally aglycan-modifying agent, increasing amounts of these agents are contactedwith the platelets prior to storing platelets at room temperature and/orexposing the platelets to a chilling temperature. The optimalconcentrations of the β-galactosidase inhibitors, the sialidaseinhibitors if used together with the β-galactosidase inhibitors, and/orglycan-modifying agent(s) that prevent cleavage of the sialic acid,prevent cleavage of β-galactose, and/or catalyze the modification of theglycan moiety are the minimal effective concentrations that preserveintact platelet function as determined by in vitro tests (e.g.,observing morphological changes in response to glass, thrombin,cryopreservation temperatures; ADP-induced aggregation) followed by invivo tests indicative of hemostatic function (e.g., recovery, survival,and shortening of bleeding time in a thrombocytopenic animal or recoveryand survival of ⁵¹Cr-labeled platelets in human subjects).

Methods of Preparing Platelet Compositions

The invention, in other aspects, provides a novel method of preparing aplatelet composition involving obtaining a population of isolatedplatelets from a donor and treating the platelets with an effectiveamount of a β-galactosidase inhibitor, or both an effective amount of aβ-galactosidase inhibitor and a sialidase inhibitor within a time perioddescribed herein. In a preferred embodiment, the novel method ofpreparing a platelet composition comprises obtaining a population ofplatelets from a donor; adding an effective amount of a β-galactosidaseinhibitor or both an effective amount of a β-galactosidase inhibitor anda sialidase inhibitor to the population of platelets and storing theresulting platelet composition for a period of time at room temperaturewithout a substantial loss of in vivo hemostatic activity. In anotherpreferred embodiment, the novel method of preparing a plateletcomposition includes obtaining a population of platelets from a donor;adding an effective amount of a β-galactosidase inhibitor or both aneffective amount of a β-galactosidase inhibitor and a sialidaseinhibitor to the population of platelets; cooling the resulting plateletcomposition to a temperature below room temperature; storing theplatelet composition for a period of time at a temperature below roomtemperature and rewarming the platelet composition back to roomtemperature without a substantial loss in vivo hemostatic activity. Infurther embodiments, the platelet composition is rewarmed slowly. Incertain embodiments, the population of platelets retains substantiallynormal hemostatic activity when transfused into a mammal. In furtherembodiments, the population of platelets when transfused into a mammal,has a circulation half-life of about 5% or greater (e.g., 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 100%, or 150%) than the circulationhalf-life of untreated platelets. In certain preferred embodiments, thetreated platelet population is suitable for transfusion into a human.

Preferred embodiments of the inventive methods of preparing a plateletcomposition as described herein encompass treating the population ofplatelets with an effective amount of a β-galactosidase inhibitor orboth an effective amount of a β-galactosidase inhibitor and a sialidaseinhibitor as described herein.

Further preferred embodiments of the inventive methods of preparing aplatelet composition, as described herein, involve treating a populationof platelets an effective amount of a β-galactosidase inhibitor or bothan effective amount of a β-galactosidase inhibitor and a sialidaseinhibitor, and further treating the population of platelets with aneffective amount of at least one glycan-modifying agent, as describedherein.

In some embodiments the invention provides for the combination of themethods of treating platelet described herein with one or more othermethods of platelet preservation known in the art. For example themethods of platelet modification provided in the present invention areuseful in combination with the methods described in, e.g., but notlimited to, the following US Patent Publication No.: 20090053198 A1, andU.S. Pat. Nos. 7,030,110; 7,029,654; 7,005,253; 6,900,231; 6,866,992;6,730,783; 6,706,765; 6,706,021; 6,693,115; 6,638,931; 6,635,637;6,566,379; 6,521,663; 6,518,310; 6,514,978; 6,497,823; 6,476,016;6,472,399; 6,420,397; 6,417,161; 6,350,764; 6,344,486; 6,344,466;6,326,492; 6,277,556; 6,245,763; 6,235,778; 6,221,669; 6,204,263;6,037,356; 5,919,614; 5,763,156; 5,753,428; 5,660,825; 5,622,867;5,582,821; 5,571,686; & 5,569,579; 5,550,108; 5,529,821; 5,474,891;5,466,573; 5,399,268; 5,376,524; 5,344,752; 5,269,946; 5,256,559;5,236,716; 5,234,808; and 5,198,357.

Kits

The present invention also provides kits that are used for plateletcollection, processing and storage, further including suitable packagingmaterials and instructions for using the kit contents. It is preferredthat all reagents and supplies in the kit be sterile, in accordance withstandard medical practices involving the handling and storage of bloodand blood products. Methods for sterilizing the kit contents are knownin the art, for example, ethylene oxide gas, gamma irradiation, and thelike. In certain embodiments, the kit may include venipuncture suppliesand/or blood collection supplies, for example a needle set, solution forsterilizing the skin of a platelet donor, and a blood collection bag orcontainer. Preferably the container is “closed”, i.e., substantiallysealed from the environment. Such closed blood collection containers arewell known in the art, and provide a means of preventing microbialcontamination of the platelet preparation contained therein. Otherembodiments include kits containing supplies for blood collection andplatelet apheresis. The kits may further include a quantity of one ormore β-galactosidase inhibitors or both a quantity of one or moreβ-galactosidase inhibitors and one or more sialidase inhibitors with orwithout the glycan-modifying agent, sufficient to modify the volume ofplatelets collected and stored in the container. In other embodiments,the kit includes a blood collection system having a blood storagecontainer wherein the β-galactosidase inhibitor agent or bothβ-galactosidase inhibitor agent and the sialidase inhibitor agent areprovided within the container in an amount sufficient to treat thevolume of blood or platelets held by the container. The quantity of theβ-galactosidase inhibitor alone, the sialidase inhibitor and theβ-galactosidase inhibitor together, or either embodiment with theoptional glycan-modifying agent will depend, in part, on the volume ofthe container. It is preferred that the β-galactosidase inhibitor, orboth the β-galactosidase inhibitor and the sialidase inhibitor, andoptionally the glycan-modifying agent be provided as a sterilenon-pyogenic solution, but any can also be supplied as a lyophilizedpowder. For example, a blood bag is provided having a capacity of 250mL. Contained in the blood bag is a quantity of the β-galactosidaseinhibitor, β-galactosidase inhibitor together with sialidase inhibitor,or a combination such that when 250 mL of blood is added, the finalconcentration of the inhibitor(s) is approximately 1200 micromolar.

Other embodiments contain different concentrations of theβ-galactosidase inhibitor, or combination of the β-galactosidaseinhibitor and the sialidase inhibitor, for example but not limited toquantities resulting in final concentrations of 10 micromolar to 10millimolar, and preferably 100 micromolar to 1.2 millimolar of theβ-galactosidase inhibitor alone, the sialidase inhibitor andβ-galactosidase inhibitor together, or with the combination of thesialidase inhibitor, the β-galactosidase inhibitor, and theglycan-modifying agent. Other embodiments use combinations of sialidaseinhibitor/β-galactosidase inhibitor with the glycan-modifying agent,e.g., to effect sialyiation or galactosylation of glycans on bloodproducts introduced into the container.

Platelet Function and Assessment of Treated Platelets

After treatment of platelets, the platelet functions can be assessedwith various in vitro methods. The recovery and survival of the treatedplatelets can be further evaluated, which are mostly performed withradioactive-labeled platelets in healthy volunteers.

“Hemostatic activity,” as described herein, refers to the ability of apopulation of platelets to mediate bleeding cessation (e.g., to form aclot). Normal hemostatic activity refers to an amount of hemostaticactivity seen in the treated platelets, that is functionally equivalentto or substantially similar to the hemostatic activity of untreatedplatelets in vivo, in a healthy (non-thrombocytopenic ornon-thrombopathic mammal) or functionally equivalent to or substantiallysimilar to the hemostatic activity of a freshly isolated population ofplatelets in vitro.

After treatment, platelets can be assessed to determine if theymaintained their function, e.g., their ability to activate and form aclot. Various assays are available for determining platelet hemostaticactivity (Bennett, J. S. and Shattil, S. J., 1990, “Platelet function,”Hematology, Williams, W. J., et al., Eds. McGraw Hill, pp 1233-12250).In an embodiment, demonstration of “hemostasis” or “hemostatic activity”can also include a demonstration that platelets infused into athrombocytopenic or thrombopathic (i.e., non-functional platelets)animal or human circulate and stop natural or experimentally-inducedbleeding. To determine hemostatic activity of platelets, laboratoriesuse in vitro tests. These tests, which include assays of aggregation,secretion, platelet morphology and metabolic changes, measure plateletfunctional responses to activation. These in vitro tests reliablyevaluate and predict in vivo hemostatic platelet function.

In an embodiment, platelets treated with compositions of the presentinvention (e.g., β-galactosidase inhibitors; combinations of sialidaseinhibitors and β-galactosidase inhibitors) exhibit a level of plateletfunction similar to that of untreated but freshly obtained/isolatedplatelets.

A test that measures the platelets' ability to clot is an aggregationassay. The platelet aggregation test uses an aggregometer to measure thecloudiness or turbidity of blood plasma. Agonists to promote clottingare used in an aggregation assay. Examples of agonists include adenosinediphosphate (ADP), epinephrine (adrenaline), thrombin, collagen, TXA2,and ristocetin. Since agonists are added to the sample in order toperform the test, the results are impacted if the donor of the sample istaking an anticoagulant. The addition of an agonist to a plasma samplecauses the platelets to clump together, making the fluid moretransparent. The aggregometer then measures the light transmissionthrough the specimen to determine the extent of the clotting by theplatelets in response to the agonist. When an agonist is added theplatelets aggregate and absorb less light and so the transmissionincreases and this is detected by the photocell in the aggregometer. Thenormal time for platelet aggregation varies somewhat depending on thelaboratory, the temperature, the shape of the vial in which the test isperformed, and the patient's response to different agonists.Establishing normal clot times and amounts of agonists for anaggregation assay can be determined by one of skill in the art.Exemplary amounts of agonist are as follows: ADP between 1 μM to 10 μM,collagen between 1 and 4 μg/mL, Ristocetin between 0.5 mg/mL and 1.5, 5mg/mL, adrenaline between 5 and 10 μM, arachadonic acid (precursor ofTXA2) about 500 μg/mL, and thrombin between 50 nmol/L and 100 nmol/L.For example, the difference between the response to ristocetin and otherproducts should be noted because ristocetin triggers aggregation througha different mechanism than other agonists. Platelets that have about 65%or greater platelet aggregation in response to adenosine diphosphate(ADP), arachidonic acid, collagen, thrombin, TXA2, epinephrine, and/orristocetin are considered platelets with normal clotting function.Accordingly, platelets treated with the β-galactosidase inhibitors (orβ-galactosidase inhibitors together with sialidase inhibitors) of thepresent invention and exhibiting about 65% or greater (e.g., about 65%to about 100%) platelet aggregation in an aggregation assay areconsidered to exhibit homeostatic activity.

Another test that measures coagulation is thrombelastography.Thrombelastography is available, for example, from HaemoneticsCorporation (Braintree, Mass.) under the trade name TEG. Inthrombelastography, a small sample of platelets (typically 0.36 mL) isplaced into a cuvette (cup) which is rotated gently through 4° 45′(cycle time 6/min) to imitate sluggish venous flow and activatecoagulation. When a sensor shaft is inserted into the sample a clotforms between the cup and the sensor. The speed and strength of clotformation is measured in various ways, and depends on the activity ofthe plasmatic coagulation system, platelet function, fibrinolysis andother factors that can be affected by illness, environment andmedications. Generally, four values that represent clot formation aredetermined by this test: the R value (or reaction time), the K value,the angle, and the MA (maximum amplitude). The R value represents thetime until the first evidence of a clot is detected. The K value is thetime from the end of R until the clot reaches 20 mm and this representsthe speed of clot formation. The angle is the tangent of the curve madeas the K is reached and offers similar information to K. The MA is areflection of clot strength. A mathematical formula determined by themanufacturer can be used to determine a Coagulation Index (CI), whichtakes into account the relative contribution of each of these 4 valuesinto 1 equation. The treated platelets of the present invention are ableto form clots, and maintain hemostasis.

Immunological Assessment of Platelet Markers/Function

Platelet function, including its ability to activate before and/or aftertreatment with the composition and also after transfusion into anindividual, can be assessed. Examples of platelet activation markersinclude P-selectin, PAC-1, GPIIb, GPIIIa, GPIb, and GPIIIa. Soluble andmembrane bound markers can be assessed to determine the state ofplatelet activation and assess homeostasis of the treated plateletpreparation. Methods that measure soluble and membrane bound plateletmarkers include several suitable assays. Suitable assays encompassimmunological methods, such as flow cytometry, radioimmunoassay,enzyme-linked immunosorbent assays (ELISA), chemiluminescence assays,and assessment with a volumetric capillary cytometry system. Any methodknown now or developed later can be used for measuring such markers.

The inventive methods use antibodies reactive with platelet markers orportions thereof. In a preferred embodiment, the antibodies specificallybind with membrane bound and/or soluble platelet makers or a portionthereof. When the antibodies bind, they inhibit the function of theprotein or marker to which they bind. The antibodies can be polyclonalor monoclonal, and the term antibody is intended to encompass polyclonaland monoclonal antibodies, and functional fragments thereof. The termspolyclonal and monoclonal refer to the degree of homogeneity of anantibody preparation. They are not intended to be limited to particularmethods of production.

In several of the preferred embodiments, immunological techniques detectplatelet marker levels by means of an anti-platelet marker antibody(e.g., one or more antibodies). An anti-platelet marker antibodyincludes monoclonal and/or polyclonal antibodies, and mixtures thereof.Labeling platelets with antibodies directed against surface membraneglycoproteins and then analyzing the binding by flow cytometry is arapid and sensitive technique for assessing homeostasis. For example,GPIIb, GPIIIa, and GPIb can be assessed using antibodies CD41, CD61, andCD42b, respectively. Elevated levels of membrane bound or solubleP-selection can indicate the extent of platelet activation and can bedetected using monoclonal antibodies S12 or W40. Antibodies fordetecting such markers can be purchased commercially or raised againstan appropriate immunogen using methods known in the art.

Any method known now or developed later can be used for measuringmembrane bound platelet markers. One method for assessing membrane boundplatelet marker levels which the invention utilizes is flow cytometry.Methods of flow cytometry for measuring platelet or membrane boundmarkers are well known in the art. (Shattil, Sanford J, et al.“Detection of Activated Platelets in Whole Blood usingActivation-Dependant Monoclonal Antibodies and Flow Cytometry,” Blood,Vol. 70, No 1 (July), 1987: pp 307-315; Scharf, Rudiger E., et al.,“Activation of Platelets in Blood Perfusing Angioplasty-damaged CoronaryArteries, Flow Cytometric Detection,” Arteriosclerosis and Thrombosis,Vol 12, No 12 (December), 1992: pp 1475-1487, the teachings of which areincorporated herein by reference in their entirety). For example, asample comprising platelets can be contacted with an antibody havingspecificity for the marker under conditions suitable for formation of acomplex between an antibody and marker expressed on platelets, anddetecting or measuring (directly or indirectly) the formation of acomplex. In an example, the level of membrane bound markers can beassessed by flow cytometry by obtaining a first and second samplecomprising platelets, contacting said first sample, serving as acontrol, with a platelet activation agonist, such as phorbol myristateacetate (PMA), ADP (adenosine diphosphate), thrombin, collagen, and/orTRAP (thrombin receptor activating peptide), under conditions suitablefor activation of platelets in said first sample, preferably for aperiod of time effective to maximally activate said platelets, andpreferably while maintaining the second sample under conditions suitablefor maintaining the endogenous platelet activation level. The methodthen involves contacting or staining the samples with a compositioncomprising an anti-platelet marker antibody, having a fluorescent label,preferably in an amount in excess of that required to bind the markerexpressed on the platelets, under conditions suitable for the formationof labeled complexes between said antibody and activated platelets. Thenone determines (detecting or measuring) the formation of complex in saidsamples, wherein the amount of complex detected indicates the extent ofplatelet activation in said second sample. In an embodiment, the amountof platelet activation in isolated platelets treated with thecomposition of the present invention and stored is similar to the amountof platelet activation from freshly obtained platelets from a donor.

In addition to using flow cytometry to measure membrane bound plateletmarkers, a radioimmunoassay can also be employed. Using aradioimmunoassay, endogenous platelet activation can be assessed by animmunobinding assay by obtaining a first and second sample comprisingplatelets, wherein each sample contains a preselected number ofplatelets; contacting said first sample with a platelet activationagonist, such as phorbol myristate acetate (PMA), ADP (adenosinediphosphate), thrombin, collagen, and/or TRAP (thrombin receptoractivating peptide), under conditions suitable for activation ofplatelets in said first sample, preferably for a period of timeeffective to maximally activate said platelets, and preferably whilemaintaining the second sample under conditions suitable for maintainingthe endogenous platelet activation level. Then the samples are contactedwith an antibody composition that is specific to the marker beingassessed. The antibody can have a radioactive label; or a binding sitefor a second antibody that has the radioactive label. The formation ofthe complex in the samples are detected, wherein the amount of complexdetected in said second sample as compared to that detected in saidfirst sample is indicative of the extent of platelet activation in saidsecond sample.

Assaying for Detection of Soluble Platelet Markers

Any method known now or developed later can be used for measuringsoluble platelet markers. In a preferred embodiment, soluble plateletmarker is determined using an ELISA assay or a sandwich ELISA assay. Fordetection of a soluble platelet marker in a suitable sample, a sample(e.g., blood) is collected, and preferably platelets are removed(partially or completely) from the sample, for example by preparation ofserum or plasma (e.g., isolation of platelet poor plasma). Samples arepreferably processed to remove platelets within a time suitable toreduce artificial increases in soluble platelet marker, such as thosedue to secretion or proteolysis from platelets. Samples can be furtherprocessed as appropriate (e.g., by dilution with assay buffer (e.g.,ELISA diluents)). Additionally, the technician can add a reagent thatstabilizes and prevents in vitro platelet activations. Examples of thesestabilizing reagents are apyrase and prostaglandin E1 (PGE₁).

To determine a measurement for soluble platelet markers using an ELISAassay in a suitable sample such as serum or platelet poor plasma (PPP),the method involves combining a suitable sample and a composition thatincludes an anti-platelet antibody as detector, such as biotinylatedanti-platelet MAb and HRP-streptavidin, or HRP-conjugated anti-plateletMab, and a solid support, such as a microtiter plate, having ananti-platelet marker capture antibody bound (directly or indirectly)thereto. The detector antibody binds to a different epitope from thatrecognized by the capture antibody, under conditions suitable for theformation of a complex between said anti-platelet maker antibodies andsoluble platelet marker. The method involves determining the formationof complex in the samples.

The solid support, such as a microtiter plate, dipstick, bead, or othersuitable support, can be coated directly or indirectly with ananti-platelet maker antibody. For example, an anti-platelet markerantibody can coat a microtiter well, or a biotinylated anti-plateletmarker Mab can be added to a streptavidin coated support. A variety ofimmobilizing or coating methods as well as a number of solid supportscan be used, and can be selected according to the desired format.

In a particularly preferred embodiment, the sample (or standard) iscombined with the solid support simultaneously with the detectorantibody, and optionally with one or more reagents by which detection ismonitored.

A known amount of soluble platelet maker standard can be prepared andprocessed as described above for a suitable sample. This standardassists in quantifying the amount of the maker detected by comparing thelevel of platelet marker in the sample relative to that in the standard.A physician, technician, apparatus or a qualified person can compare theamount of detected complex with a suitable control to determine if thelevels are elevated.

Typical assays for platelet markers are sequential assays in which aplate is coated with first antibody, plasma is added, the plate iswashed, second tagged antibody is added, the plate is washed, and boundsecond antibody is quantified. However, binding kinetics revealed thatin a simultaneous format, the off-rate of the second antibody wasdecreased and the assay was more sensitive. Thus, a simultaneous formatin which the solid support is coated with a capture antibody, and plasmaand detector antibody are added simultaneously, can achieve enhancedsensitivity and is preferred.

A technician, physician, qualified person or apparatus can compare theresults to a suitable control such as a standard, levels of one or moreplatelet markers in normal individuals, and baseline levels of theplatelet markers in a sample from the same donor. For example, the assaycan be performed using a known amount of a platelet marker standard inlieu of a sample, and a standard curved established. One can relativelycompare known amounts of the platelet marker standard to the amount ofcomplex formed or detected.

Storage lesions can be assessed to determine the health of a plateletand its ability to activate and form a clot. Storage lesions includemorphological and molecular changes to platelets upon storage at orbelow room temperature. One of the first visible effects of plateletimpairment is the irreversible loss of the discoid morphology towards aspherical shape, and the appearance of spiny projections on the surfacedue to calcium-dependent gelsolin activation andphosphoinositide-mediated actin polymerization. Certain morphologicalchanges induced in platelets can be readily observed under a microscope.A loss in shape is accelerated at low temperatures and particularly whenplatelets are exposed to temperatures lower than 20° C. In addition toincreased modifications in shape, notable increases occur inintracellular calcium levels and in the degree of actin polymerization.Moreover, stored platelets secrete alpha granule and lysosomal contents,which can be assessed immunologically, as described herein, andreorganize the microtubule coil lying under the plasma membrane throughdepolymerization processes. Accordingly, storage lesions that occur ator below room temperature can readily be measured by methods known inthe art and described herein to quantify the effectiveness of theinventive platelet compositions and related methods. The standard is tocompare the quality of the platelet storage solution of the presentinvention to the quality of platelet storage solutions without aβ-galactosidase inhibitor or to the quality of platelet storagesolutions without a β-galactosidase inhibitor and a sialidase inhibitor.Accordingly, platelets treated with the composition of the presentinvention maintain shape and function that is at least similar to orbetter than platelets not stored in the PPS of the present invention(e.g., stored in a known platelet storage solution such as INTERSOL®solution (Fenwal) and SSP+™ solution (MacoPharma)).

EXEMPLIFICATION Example 1 Human Platelets

Prolonged storage at and below room temperature resulted in sialic acidloss and increased sialidase (neuraminidase) activity for human_platelets

Loss of Platelet Sialic Acid During Prolonged Storage UnderRefrigeration:

Platelets were stored at 4° C. in the absence or presence of 1.2 mMnucleotide sugars and the total sialic acid was quantified. Theplatelets were centrifuged, thoroughly washed, and resuspended in 140 mMNaCl, 3 mM KCl, 0.5 mM MgCl₂, 5 mM NaHCO₃, 10 mM glucose and 10 mMHEPES, pH 7.4. Aliquots of the resuspended platelets were lysed withRIPA buffer (Cell Signaling Technology) for protein quantification usingPierce BCA Protein Assay Kit, or processed to quantify platelet sialicacid using QUANTICHROM® Sialic Acid Assay Kit per the manufacturer'sinstructions (BioAssays Systems). The assay kit uses an improved Warrenmethod in which sialic acid is oxidized to formylpyruvic acid, whichreacts with thiobarbituric acid to form a pink colored product. Theabsorbance at 549 nm is directly proportional to sialic acidconcentration, which in the test sample can be calculated from a linearstandard curve obtained from sialic acid standards per themanufacturer's instructions. Fresh platelets contain ˜10 μg (i.e.,approximately 10 micro-grams) of sialic acid per mg of platelet protein.Prolonged storage under refrigeration resulted in great loss of plateletsialic acid (Day 5_a, Donor A, ˜35%; Donor B, ˜25%), compared with freshplatelets (Day 0), normalized to 100%. However, the loss of sialic acidwas slowed in donor B platelets by the presence of CMP-sialic acid andUDP-gal (B_Day_5_b) in the stored platelets, the donor sugar requiredfor resialylation (FIG. 2). UDP-gal alone had no effect (Day 5_c). It isnoted that the platelets from Donor B with less sialic acid loss hadless initial sialidase surface activity than those from Donor A (Seebelow, FIG. 3B).

Sialidase Activity During Platelet Storage:

Human platelets express surface-exposed sialidases. Sialidase activityis a particular concern since it is presumably responsible for the lossof platelet sialic acid during storage. Therefore, in addition to thedirect analysis of sialic acid content, quantification of the totalplatelet sialidase activity and surface sialidase activity duringstorage are critical to understand the mechanism of sialic acid loss.Furthermore, sialidase activity may hinder an attempted resialylationapproach. A determination of the nature of the sialidases in fresh andstored platelets is important. Shown herein is a reliable and sensitivefluorometric assay method for platelet sialidase activity using4-methylumbelliferyl-α-D-N-acetylneuraminic acid (4-MU-NeuAc) as asubstrate. Cleavage of the substrate by sialidase released sialic acidand methylumbelliferone (MU), upon termination with Na₂CO₃, wherein thelater was read at λex/em=355/460 nm. Sialidase activity was measured innon-permeabilized or permeabilized platelets. FIG. 3A shows that intactfresh platelets do not contain significant surface sialidase activity.In contrast, abundant sialidase activity, including both surface andintracellular sialidase activities, was measured in permeabilized freshplatelets. Further analysis indicates that surface sialidase activity offresh platelets varies among donors (FIG. 3B, donor A and B), butincreased platelet sialidase activity upon cold storage was observed inall cases including Donor A and B (FIG. 3C).

The detection of increased platelet sialidase activity uponrefrigeration and its absence in the storage media (not shown) suggestedthat cool temperatures may increase the surface exposure ofsialidase(s). To test this assumption, the sialidase exposure onplatelets was examined by immunofluoresence. Four human sialidases havebeen identified, Neu1, Neu2, Neu3 and Neu4. Neu1 is a lysosomal enzyme;Neu2 is a cytosolic sialidase; Neu3 is a plasma membrane sialidase,wherein its activity is specific for gangliosides; and Neu4 is a novelhuman luminal lysosomal enzyme. Neu1, Neu2, Neu3, and Neu4 share highdegrees of similarity and amino acid blocks of highly conservedresidues. However, these sialidases are different from one another interms of subcellular localization, substrate preference in vitro, andtissue distribution. Neu1 is a lysosomal sialidase that is presumed tohave a narrow substrate specificity. The natural substrate for thisenzyme is unknown and activity has thus only been reported on artificialsubstrates such as 4-MU-NeuAc and nitro-phenyl-NeuAc, but not ongangliosidases, fetuin, or sialyllactose. Neu2 is a cytosolic enzymewith wide substrate specificity. Neu3 is a plasma membrane-boundsialidase, originally described as ganglioside sialidase. Neu3preferentially hydrolyses gangliosides, although glycoproteins,4-MU-NeuAc, sialyllactose, etc. are also hydrolysed. Lysosomal Neu1 andsurface-bound Neu3 (antibodies are commercially available) were thefocus of the current studies. As shown in FIG. 4, Neu3 can readily bevisualized on the surface of fresh platelets and its expression is notaffected by refrigeration. In contrast, Neu1 only demonstrated weaksurface exposure on fresh platelets, consistent with its subcellularlocalization in an intracellular lysosomal granule. However, uponrefrigeration for 48 h, its surface exposure is greatly increased. Thedata demonstrates that Neu1 is at least partially responsible for theplatelet surface sialidase activity increase during refrigeration.

In summary, platelet storage under refrigeration promotes plateletsurface sialic acid loss and increases platelet surface sialidaseexpression. Similar findings were also made for RT-stored platelets (notshown).

Example 2 Mouse Platelets

Sialidase activity increases during cold storage of mouse platelets andthe sialidase inhibitor DANA increases mouse platelet survival in vivo.

Mouse Platelet Sialidase Activity Increases Following 48 h Cold Storage:

We have determined sialidase surface activity in isolated, intact, freshmouse platelets and following cooling and rewarming using Amplex RedNeuraminidase (Sialidase) Assay Kit (Molecular probes, Eugene, Oreg.,USA). Mouse platelets (2×10⁹) maintained at room temperature orrefrigerated for 48 h were isolated and suspended in the providedreaction buffer (0.5 M Tris-HCl, pH 7.2 and 1 mM CaCl₂). Plateletderived sialidase activity was measured over 2.5 h at room temperature.FIG. 5 shows that sialidase activity substantially increases followingplatelet storage in the cold (4° C.) compared to fresh room temperatureplatelets (RT). Critically, sialidase activity is not plasma derived, asplatelets were extensively washed prior to sialidase activity assays. Asa control, sialidase activity (Clostridium perfringens (Component H))was measured over the same time period (inset). Neu1 surface expressionis increased by 3.5 fold on stored platelets as determined by flowcytometry using anti-Neu1 specific antibodies (not shown).

Fetuin as a Competitive Sialidase Substrate During Platelet Storage:

Fetuin (1 mg/mL) was added to mouse platelet rich plasma prior to coldstorage or to fresh platelets at room temperature and β-galactoseexposure measured by flow cytometry using FITC conjugated RCA-1-lectin,a lectin specific for exposed β-galactose. Addition of fetuin greatlyinhibits sialic acid hydrolysis during platelet storage, preventingRCA-1 binding. Fetuin addition has no effect on RCA-1 binding to freshplatelets (FIG. 6). These results show that sialidase activity increasesduring platelet cold storage, presumably mediating sialic acidhydrolysis.

The Sialidase Inhibitor DANA Increases Platelet Life Span In Vivo:

The quantification of sialic acid was determined in freshly isolatedplatelets and long-term stored platelets using a Sialic AcidQuantification Kit (Sigma, St. Louis Mich., USA). The Sialic AcidQuantification Kit determines total N-acetylneuraminic acid (sialicacid) following the release from glycoconjugates usingα2-3,6,8,9-neuraminidase to cleave all sialic acid linkages, includingbranched sialic acid. Results show that 2×10⁹ freshly isolated mouseplatelets (˜2.5 mg protein) contain ˜3 μmol sialic acid. Followinglong-term storage, platelets lose >50% of their sialic acid content (notshown).

It had been previously postulated that sialic acid normally coversβ-galactose residues and permits platelet survival. These results showthat normal platelet survival is regulated by hepatocyte ASGP receptor,independent of macrophages. Surface sialic acid is normally hydrolyzedby sialidases. These studies then addressed whether inhibition ofsialidase activity in vivo has an effect on platelet survival. Mouseplatelets have prolonged survival after injecting mice with the specificsialidase inhibitor, sodium salt of2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA). Mice were injectedwith 100 mM DANA or PBS (phosphate buffered saline) as a control, afterin vivo platelet biotinylation. Inhibition of sialidase activity withDANA increases the survival of biotinylated platelets (DANA) compared tobiotinylated platelet survival in control mice (Control) (FIG. 7). Theseresults indicate that inhibition of neuraminidase activity in vivoprolongs platelet survival. However, the effect may not be plateletspecific. As shown, the recovery and survival of fresh platelets aresignificantly enhanced in Asgr-1 or Asgr-2 deficient mice (Sorensen etal., Blood, 2009, Vol. 114, pgs 1645-1654) revealing that the hepatocyteAshwell-Morell receptors routinely survey the platelet surface forβ-galactose exposure. Taken together, these data indicate that plateletslose sialic acid while circulating, possibly due to sialidase activity,representing a new clearance mechanism for senile platelets.

Example 3 The Role of Sialylation/Desialylation in Defining theCirculatory Lifetimes of Platelets

Human Platelets Produce Neu1 and Neu3 and Release Neu1 into Plasma:

The studies herein address two novel mechanisms that contribute toincreases in the clearance of platelets that occur upon storage. Thefirst platelet clearance mechanism, which is induced rapidly byrefrigeration in the absence of plasma, is mediated when GlcNAc residueson the N-linked glycan of GPIbα become exposed and are recognized by thelectin domain of the αMβ2 receptor on liver phagocytes. The secondclearance mechanism, induced by long-term platelet storage in plasma inthe cold, is of slow onset and occurs when GPIbα is desialylated andrecognized by the ASGP receptors on both liver hepatocytes andmacrophages. Recent data unveils an unexpected role for endogenoussialidases and glycosyltransferases (GTs) in modulating the circulatorylife times of normal platelets. In addition, as demonstrated herein,platelets express GTs and sialidases on their surfaces and secrete both.The convergence of these two mechanistic pathways strongly suggests thatplatelets have an inherent capacity to self-regulate survival in bloodby renewing the glycans of their surface glycoproteins and that plateletlifetimes can be modulated in either positive or negative directions bycarbohydrate addition or removal machinery, respectively. The glycanstructures promoting platelet circulation or clearance are thus ideallysuited for therapeutic manipulation by sialidases or GT activity (SeeFIG. 8).

Lysates of human platelets were subjected to SDS-PAGE and immunoblottingusing antibodies specific for Neu1 and Neu3 (provided by Dr. N.Stamatos, Univ. of Maryland). FIG. 9 shows that human platelets containboth Neu1 and Neu3. FIG. 10 shows that human platelets release Neu1 intoplasma after 24 hours of storage in the cold, indicating that releasedNeu1 could mediate the removal of surface sialic acid from plateletGPIbα. As predicted from FIG. 10 and FIG. 4, sialidase activityassociated with platelet surface increases with the time of cooling.

Human Platelets Express Glycosyltransfereses and Release them intoPlasma Upon Activation:

Glycosyltransfereses (GTs) are expressed on platelets and packagedinternally into a secretory compartment. Platelets have a surfaceassociated β4gal-T (β4Gal-T1) that catalyzes the coupling of Gal in aβ1-4 linkage to exposed N-acetylglucosamine (GlcNAc) residues on theN-linked glycans of GPIbα, improving short-term cooled mouse plateletcirculation (Hoffmeister K M, Josefsson E C, Isaac N A, Clausen H,Hartwig J H, Stossel T P. Glycosylation restores survival of chilledblood platelets. Science. 2003 Sep. 12; 301(5639):1531-4). The nature ofthis glycosylation machinery is becoming increasingly evident from thedata provided herein. For example, platelets were paneled withantibodies to determine which enzymes are expressed. Human plateletlysates were displayed by SDS-PAGE and immunoblotted against antibodiesthat recognize GTs. Cross-reactive proteins are present against 3GalNAc-Ts, a Gal-T, and a Sial-T (FIG. 11A).

The presence of internal GT stores suggests that platelets might moveGTs to their surface upon activation. The amount of each GT isoformassociated with either resting platelets or activated platelets wasassessed, as was the amount released into the corresponding medium.Resting platelets were maintained at 37° C. or treated with 25 μM TRAPfor 5 min. Maximal release was observed after 1 minute. Enzymaticactivity remaining in the 800×g pelleted platelets (P) or was releasedinto the media (M). The media was clarified at 100,000×g for 90 minprior to activity measurements. FIG. 11B shows that ˜93% of the total GTactivity associates with resting platelets, as collected bycentrifugation (P), although a small portion of the activity is releasedinto the bathing medium (M). However, following activation of plateletswith 25 μM thrombin receptor activating peptide (TRAP) for 5 min, theamount of cell associated activity drops and ˜50% of the total GalNAc-,Gal-, and Sial-T activities are released into the medium.Ultracentrifugation did not remove enzymatic activity from thesupernatant, excluding the possibility that the secreted activityresides in platelet microparticles. Hence, GTs are packaged withinplatelets in a secretory compartment. The nature of this internal GTcompartment was also addressed. Immunofluorescent labeling of fixed andpermeabilized platelets with antibodies directed towards certainselected GTs, or the well-characterized Golgi matrix protein GM130,revealed internal staining of 2-5 granular structures per platelet (notshown). Hence, platelets contain abundant amounts of GTs and sialidasesand the ability of platelets to circulate depends on having GPIbα in amaximally sialylated state.

Endogenous Active Platelet Sialyltransferases Incorporate Sialic Acidinto GPIbα:

Endogenous resialylation was studied by following the fate of i. v.injected fluorescent-CMP-sialic acid (FITC-SA) in mouse platelets. Afterthe injection, platelets were isolated and analyzed for theincorporation of fluorescence by flow cytometry (FIG. 12) and bydetermining the extent to which the fluorescent-tag was incorporatedinto mouse (not shown) and human GPIbα (FIG. 12) by SDS-PAGE andimmunoblotting analysis. Similar results were obtained using ¹⁴CCMP-sialic acid, as shown in FIG. 12. FITC labeled CMP-SA (FITC-SA) orFITC alone (FITC) were injected into wild type mice. After 1 hour, themice were bled and FITC incorporation into platelets was determined byflow cytometry. Isolated human platelets were incubated with FITC (F),FITC-SA, or left untreated (−). Resting (Rest) and TRAP (TRAP) activatedplatelets were subjected to immunoblotting using anti-FITC (FITC),-GPIbα, -αIIb or -von Willebrand factor (vWf) antibodies. Actin is shownas a loading control.

Proteolysis of GPIbα and GPV by the Metalloprotease TACE (ADAM17) is notRequired to Initiate Platelet Clearance after Desialylation:

During room temperature platelet storage or platelet storage underrefrigeration, the loss of GPIbα and GPV is observed. In contrast, otherplatelet receptors, such as GPIX, GPIbβ, GPVI, or β3 remain unchangedfollowing platelet storage independent of the storage temperature (FIG.13). TACE mediates proteolysis of GPIbα and GPV during plateletrefrigeration as shown by inhibition of TACE using the metalloproteaseinhibitors GM6001 or platelets deficient for TACE (FIG. 14).Surprisingly preservation of receptor loss during platelet refrigeratedstorage does not prevent refrigerated platelet clearance (FIG. 14).Removal of sialic acid from TACE deficient platelets diminishes plateletcirculatory lifetime (FIG. 15C). This demonstrates that proteolysis ofGPIbα or GPV is not required to initiate platelet clearance afterdesialylation. Platelets were isolated from TACE activity deficient miceand treated with sialidase for 15 min at 37° C. (+Neu) or left untreated(-Neu). Fluorescently (CMFDA) labeled platelets (2×10⁸) were injectedinto wild type mice and their circulation times were determined.Importantly, no differences in surface vWf receptor expression wereobserved in sialidase (Neu) treated and untreated TACE^(−/−) plateletswhen measured by flow cytometry (FIG. 15B). In contrast, after sialidasetreatment, β-galactose exposure increased by ˜5 fold as determined usingRCA I and ECL lectins (FIG. 15A)).

Example 4 Surface Sialic Acid Prevents Loss of GPIbα and GPV DuringPlatelet Storage and Rescues In Vivo Survival of Mouse Platelets

Platelet processing and storage are associated with platelet lesion(e.g., shape change, activation, release reaction, and apoptosis), whichis partially due to loss of surface receptors. Surface sialic acid isconsidered to be a key determinant for the survival of circulating bloodcells and glycoproteins. However, its role in platelet receptor loss andplatelet survival is unclear. In this study, the relationship betweensurface sialic acid and platelet receptor loss was investigated in vitroand in vivo.

Removal of Sialic Acid from Platelet vWf Receptor Stimulates GPIbα andGPV Shedding:

Incubation of mouse platelets with increasing concentrations of thebroad spectrum A. ureafaciens α2-3,6,8-sialidase increased surfaceβ-galactose exposure, but not β-GlcNAc, as detected by lectin bindingassays in the flow cytometer (FIG. 16). FIG. 17 presents progressiveloss of surface of surface GPIbα and GPV in conjunction with decrease insialic acid content (p<0.05). GPIbα receptor expression was followedwith multiple anti-GPIbα antibodies to exclude the possibility thatdesialylation altered antibody binding to GPIbα. We detected a ˜6 foldincrease of terminal β-galactose, but not β-GlcNAc, following treatmentwith 5 mU sialidase. β-Galactose exposure was completely inhibited by ofthe competitive sialidase inhibitor DANA (FIG. 18). Sialidase treatmentdid not affect the expression of surface GPIX-receptor or integrinαIIbβ3 (p >0.05) (FIG. 19). Critically, addition of the competitivesialidase inhibitor DANA prevented all GPIbα and GPV shedding (FIG. 19),consistent with the hypothesis that sialic acid loss primes GPIbα andGPV for metalloprotease-mediated shedding. FIG. 20 confirms the flowcytometry data shown in FIG. 19 by using immunoblot analysis of totalplatelet lysates, platelet supernatants, and corresponding plateletpellets with or with addition of neuraminidase and DANA. In support ofthis notion, FIG. 21 shows that fresh platelets treated with sialidaseare cleared rapidly from the circulation in a process prevented by DANAaddition to the storage buffer. Importantly, addition of DANA preservedreceptors expression of room temperature stored mouse platelets (FIG.22) and platelet survival (not shown).

Desialylation is Required for TACE-Mediated GPIbα and GPV Shedding:

To confirm that desialylated GPIbα and GPV are better TACE substratesthan the sialylated forms, platelets were treated with recombinant TACE(rTACE) in the presence or absence of DANA. Platelets treated with rTACEreleased 47%±6 and 18%±12 of their GPIbα and GPV (p<0.05), respectively(FIG. 25), but negligible amounts of their GPIX and α_(IIb)β₃ (p >0.05)(not shown). Receptor shedding by rTACE, but not rTACE activity (notshown) was completely prevented by DANA (FIG. 25). Addition of the MMPinhibitor GM6001 to sialidase-treated platelets did not preventβ-galactose exposure, e.g. loss of sialic acid (FIG. 23), but inhibitedreceptor shedding by rTACE (FIG. 24) (p<0.05). β-galactose exposureinduced by sialidase increased 7-fold in the presence of GM6001 andrTACE (FIG. 23), showing that GM6001 has no effect on sialidase activitybut completely inhibits rTACE and endogenous metalloprotease function.Hence, the data show that desialylation of GPIbα and GPV is a likelyprerequisite for TACE-mediated receptor shedding in the cold and supportthe concept that TACE cleavage of GPIbα depends on prior sialidaseactivation.

Example 5 Bacterial Contamination/Proliferation in Platelet ConcentratesLeads to Formation of Excessive Free Sialic Acid in the Storage Media

In hospitals and blood centers, platelets are stored at roomtemperature. To reduce the risk of bacterial growth and iatrogenicinfections after transfusion, platelet shelf life is limited to 5 daysin the United States. Platelets cannot be stored in a similar manner tored blood cells (RBC) under refrigeration with less risk for bacterialgrowth and transfusion related infections. Refrigerated platelets arerapidly cleared from the recipient's circulation, despite improved invitro function. Refrigeration of platelets irreversibly clusters theplatelet glycoprotein Ibα (GPIbα) complex, leading to rapid plateletclearance when infused through lectin-mediated pathways.

Storage of platelets for transfusion at room temperature promotesbacterial growth in bacterially contaminated (unsterile) platelets. Manybacteria are able to interact with platelets and induce plateletaggregation by direct interaction between a bacterial surface proteinand a platelet receptor or an indirect interaction where plasma proteinsbind to the bacterial surface and subsequently bind to a plateletreceptor. See FIGS. 1A-C. Bacteria secrete a variety of biologicalactive substances into their local milieu. Secreted proteins areparticularly important in bacterial pathogenesis. These proteins have arange of biological functions ranging from host cell toxicity to moresubtle alterations of the host cell for the benefit of the invader. Inbacterial contaminated platelet products, the bacterial-derived productscan be capable of triggering platelet activation or causing damage tothe platelets. Many bacteria-secreted hydrolases such proteases andglycosidases (i.e sectreted enzymes) contribute to the bacterialvirulence or are thought to play a role in promoting bacterial growth asnutrients. In platelet products, enzymes secreted by contaminatingbacteria can truncate platelet glycans and/or accelerate plateletreceptor shedding. Platelets are especially susceptible to sialidaseactivity (sialic acid hydrolysis) since they are heavily decorated withglycans terminated by sialic acid. Sialidase-mediated loss of sialicacid residues will result in clearance of the desialylated platelets bythe asialo-glycoprotein receptor (ASGR) of liver hepatocytes upontransfusion. The presence of sialidase-producing bacteria in plateletproduct will be particularly detrimental to platelets. In addition,after the loss of sialic acid, asialoglycoconjugates may becomesubstrates for the additional bacterial glycosidases. Subsequent releaseof underlying glycans will generate nutrients that will enhancebacterial proliferation and generate ligands for bacteria-plateletinteractions.

Although it is well-known that bacterial contamination in plateletproducts can lead to transfusion-related sepsis and platelet activationthrough bacteria-platelet interactions, the presence ofsialidase-producing bacteria in platelet products and their potentialimpact on platelet quality have neither been recognized nor studied. Itis expected that the presence of sialidase-producing bacteria inplatelet products desialylates sialylglycoproteins on platelets and inplasma and increases the free sialic acid concentration in the storagemedia.

Materials and Methods:

One bag of platelet concentrate (Research Blood Components, Boston,Mass.) was aseptically split into two 50-mL Falcon tubes. ProstaglandinE1 (PGE1, Sigma-Aldrich) was added to 1 μg/mL and the samples werecentrifuged for 20 min at 200×g to sediment contaminated red cells. Thesupernatant (purified platelet concentrate, PC) was removed from thecontaminating RBC and pooled in a new 50 mL falcon tube. The purified PCwas then split, providing identical products for storage at 4° C. androom temperature (RT), respectively. All steps were executed underaseptic conditions. On Days 0, 8, and 13 of storage, aliquots from eachstorage condition were removed and visually inspected for color changecaused by bacterial growth at the time of sampling. The samples werecentrifuged for 10 min at 1000×g. The resultant supernatants (plateletpoor plasma, PPP) were further centrifuged for 10 min at 10,000×g, 4° C.The supernatants from the second spin (platelet-free plasma, PFP) wereanalyzed for free sialic acid using QUANTICHROM® Sialic Acid Assay Kit(BioAssay Systems, Hayward, Calif.) according to the manufacturer'sinstructions.

Results:

Color change was readily visible on Day 8 and 13 in the PC sample storedat room temperature, suggesting the proliferation of “naturally” (asopposed to spiked) occurring bacteria under these conditions. No visiblecolor change was noticed in the 4° C. stored samples.

The free sialic acid (FSA) in fresh PRP and PFP, and PFP recovered fromstorage samples were measured and the results are shown in FIG. 26.Although human plasma contains high concentrations of total sialic acid(1-2 mM), the amount of FSA in fresh PC or PFP is only ˜4 accounting forless than 0.5% of total sialic acid. The FSA level remained unchangedduring 8-day storage at 4° C. and increased by 1.4 fold during thesecond week (day 13) of 4° C. storage (dashed line). This data showsthat under the condition that bacterial growth is retarded, the plateletsialic acid loss due to the endogenous platelet sialidase is minimal. Incontrast, during storage at RT, FSA increased by ˜3-fold on Day 8 and˜9-fold on Day 13. The rapid increase of FSA in the RT-stored samplecannot be solely attributed to action of the endogenous plateletsialidase. It is likely the result of exogenous sialidase released bycontaminating bacteria. The data also shows that the contaminatingbacteria are sialidase-producing bacteria.

Conclusion:

Sialidase-producing bacteria are potentially present in all plateletproducts. The bacterial sialidase can desialylate platelets,compromising their biological functions.

Example 6 Bacterial Proliferation in Platelet Product can be Inhibitedby Sialidase Inhibitor

Sialidases play important role in pathogenicity and nutrition ofsialidase-producing bacteria. Sialic acid occupies the terminal positionwithin glycan molecules on the surfaces of many vertebrate cells, whereit functions in diverse cellular processes such as intercellularadhesion and cell signaling. Pathogenic bacteria have evolved to usethis molecule beneficially in at least two different ways: 1) they cancoat themselves in sialic acid, providing resistance to components ofthe host's innate immune response, 2) or they can use it as a nutrient.Sialic acid itself is either synthesized de novo by these bacteria orscavenged directly from the host. Our discovery of the presence ofsialidase-producing bacteria as contaminants in platelet productsuggests a novel approach of inhibiting bacterial growth in plateletproducts by inhibiting sialidase activity with sialidase inhibitors.

Sialidase inhibitors are not new to the pharmaceutical industry. Theinfluenza virus medicines Tamiflu and Relenza inhibit the influenzavirus sialidase, which is required for spreading of the virus frominfected cells. However, they have not been used in platelet products.

Conventionally, platelets are suspended in 100% plasma. Although plasma(rather whole blood) is the natural medium of platelets in vivo, itmight have deleterious effects on platelets during storage, becauseplasma enzymes such as proteases can damage platelet membranes. Astorage solution that can maintain platelet function as well or betterthan plasma is desirable, in part to make plasma available for otherpurposes, but especially to mitigate transfusion-related adversereactions, such as TRALI. Therefore, much attention has been devoted toplatelet protection solutions with satisfactory platelet preservationcapacity with low residual plasma.

Platelet additive solutions (PASs) were first developed in the 1980s,and continue to be improved until today. The use of PASs as replacementfor plasma has a number of benefits, both for the quality of theplatelet concentrates and for the patients. The growth kinetics of modelbacteria in platelets stored in a 35%:65% ratio of plasma to INTERSOL®solution (30 mM sodium phosphate, 10 mM sodium citrate, 30 mM sodiumacetate and 70 mM sodium chloride, pH 7.4) where initial bacterialconcentrations are 0.5 to 1.6 CFUs/mL have been studied. The more rapidinitiation of log-phase growth for bacteria within a PAS storageenvironment resulted in a bacterial concentration up to 4 logs higher inthe PAS units compared to the plasma units at 24 hours. This may presentan early bacterial detection advantage for PAS-stored platelets.

To increase the formation of planktonic bacteria, thereby improving thesensitivity of the bacterial detection, platelet storage studies wereperformed in a mixture of PAS (INTERSOL® solution) and plasma (80:20).Many bacterial detection methods are available. We used SLP Reagent Set(297-51501, Wako Chemicals USA), containing silkworm larvae plasma (SLP)and 3,4-dihydrophenylalanine (DOPA), reconstituted according tomanufacturer's instruction and stored as 100 μL aliquots at -80° C. Whena sample is mixed with SLP reagent, peptidoglycan derived from the cellwall of Gram-positive and Gram-negative bacteria in the sample initiatesa series of reactions including activation of multiple serine proteasescalled prophenoloxidase (proPO) cascade. The phenoloxidase (PO) producedin the cascade reactions oxidizes the substrate in the SLP reagent,3,4-dihydrophenylalanine (DOPA), to form melain (dark blue). Thebacteria concentration in the test sample is inversely proportional tothe onset time of color development: shorter time=higher concentrationof bacteria; longer time=lower concentration of bacteria.

Materials and Methods:

One bag of a platelet concentrate was split to two 50-mL Falcon tubes,PGE1 was added. After centrifugation at 900×g for 10 min, 80% of thesupernatant (PPP) (relative to the total volume) was removed, andreplaced with equal volume of platelet protection solution. The plateletwas thoroughly re-suspended. The platelet suspensions were pooled andsplit to 4 aliquots in 15-mL Falcon tubes. DANA (1 mM) in PBS was addedto two tubes while only PBS was added to the other tubes. One pair ofsamples with and without DANA was stored at 4° C. The second pair waskept at RT (22° C.-24° C.). Aliquots of 1.0 mL were removed on Day 0 andDay 9, and immediately pelleted (5 min, 15,800×g). The supernatants werediscarded, and the pellets, containing platelets and bacteria, werestored at −80° C. until use. All experimental steps were carried outunder aseptic conditions.

The pellet containing both platelets and bacteria, recovered from 1-mLaliquots sampled at different time points, was re-suspended in 100 μL of0.1 M NaOH, and heated for 10 min at 70° C. After brief cooling, thesolution was neutralized with 135 μL of 80 mM MES. The reaction mixtureswere clarified by centrifugation (5 min at 15,800×g). Aliquots of 10 μLof the supernatant were mixed with equal volumes of SLP reagent,reconstituted from the components in the SLP kit following themanufacturer's instructions. The samples were left on the bench, andcolor development was monitored. The time of color detection (TOCD) wasrecorded.

Results:

The results are shown in FIG. 27. Selected photographs taken during theanalysis of Day 9 samples are shown in FIG. 27, panels A-C. Light, butvisible, color development was observed after 15 min for RT-storedsample without DANA, suggesting the highest bacterial concentration inthis sample. TOCD was extended to 34 min in the presence of DANA (#3,FIG. 27, panel B). Not surprisingly, bacterial growth is greatlyinhibited at low storage temperatures, TOCD in 4° C.-stored samples(FIG. 27, panel C) (<45 min) was increased compared to TOCD in RT-storedsample in the absence or presence of DANA. Its TOCD at 4° C. is furtherextended in the presence of DANA (˜50 min, FIG. 27 panel C).Quantitative data is shown in FIG. 27, panel D.

Conclusion:

Sialidase inhibitor DANA can effectively inhibit the bacterial growthduring platelet storage. Although the nature of the bacteria is unknown,they are likely sialidase-producing bacteria. In addition, it wasobserved that the contaminating bacteria are not completely dormant at4° C.

Example 7 DANA Inhibits Bacterial Proliferation in Stored MousePlatelets and Improves the Survival and Recovery of Mouse Platelets InVivo

Mouse platelets have a life-span of approximately 4-5 days, considerablyshorter than human platelets (8-10 days). They are also much less stablethan human platelets when stored at room temperature or 4° C. Themechanism of the rapid deterioration in vitro of mouse platelet is notwell understood, however it is possible that mouse platelet storage isaffected by bacterial contamination due to a lack of aseptic plateletprocurement protocol, in contrast to the collection of human platelets.To date, it remains unclear if potential bacterial contaminationcontributes to the rapid deterioration of mouse platelets.

Materials and Methods:

Mouse blood was obtained from anesthetized mice using 3.75 mg/g ofAvertin (Fluka Chemie, Steinheim, Germany) by retro-orbital eye bleedinginto 0.1 volume of Aster-Jandl anticoagulant and centrifuged at 300×gfor 8 min at RT to obtain platelet rich plasma (PRP). Platelets wereseparated from plasma by centrifugation at 1200×g for 5 min and washedtwice in 140 mM NaCl, 5 mM KCl, 12 mM trisodium citrate, 10 mM glucose,and 12.5 mM sucrose, 1 μg/mL PGE1, pH 6.0 (platelet wash buffer) bycentrifugation. Washed platelets were re-suspended at a concentration of1×10⁹/mL in 140 mM NaCl, 3 mM KCl, 0.5 mM MgCl₂, 5 mM NaHCO₃, 10 mMglucose and 10 mM HEPES, pH 7.4 (platelet resuspension buffer), labeledwith 5 μM 5-chloromethylfluorescein diacetate (CMFDA) for 15 min at 37°C. Unincorporated dye was removed by centrifugation and plateletssuspended in plasma. DANA, sialyllactose, and glucose (as a nutrient)were added to final concentrations of 0.5, 0.5 and 8 mM, respectively,from their corresponding PBS stock solutions. Only PBS was added to thecontrols. The platelet suspensions were stored at 4° C. or RT for 48 h.After 48 h, the stored platelets were transfused by retro-orbitalinjection of 3×10⁸ platelets in 200 μL. Following transfusion, blood wascollected by retro-orbital eye bleeding at time points of 5 min, 2, and24 h. The percentage of CMFDA positive platelets in PRP was determinedby flow cytometry.

Results:

All samples were visually inspected for evidence of bacterialcontamination. Severe plasma color bleaching was observed for the roomtemperature-stored platelet in the absence of DANA, suggesting bacterialgrowth. No visible change was noted under all other conditions. Therecovery and survival of mouse platelet, stored for 48 h at RT in thepresence of platelet preservatives is greatly improved compared withstored platelets lacking preservatives (FIG. 28). Mouse plateletsdeteriorate rapidly when stored at RT, which is clearly shown in FIG.29. Importantly, in the presence of DANA, sialyllactose, and glucose inthe storage media approximately 5-fold more platelets were recovered(FIG. 29) compared to control samples.

Conclusion:

Sialidase inhibitor DANA is capable of effectively preserving mouseplatelets from deterioration during storage and greatly improving therecovery and survival of transfused platelets.

Example 8 Preservation of Mouse Platelets in the Presence of DifferentConcentrations of DANA

DANA is a potent, broad-spectrum sialidase inhibitor against viral,bacterial and mammalian sialidases with Ki in the low μM range. It isused routinely at 1 mM in all our studies. It is expected that itsconcentration can be dramatically lowered while maintaining its efficacyagainst the bacteria-caused deterioration of stored mouse platelets.

Materials and Methods:

Mouse platelets were isolated as described in Example 3, re-suspended inplatelet resuspension buffer and split to four aliquots. Glucose wasadded 8 mM to all samples, and DANA was added to final concentrations of0, 0.1, 1.0, and 10 mM, respectively, both from 100 mM stock solutionsin PBS. The samples were incubated for 30 min at 37° C., centrifuged andsupernatants removed. The platelets re-suspended in plasma. DANA andglucose were restored to their initial concentrations. The plateletsuspensions were stored at RT. After 48 h, platelets under each storagecondition were counted by flow cytometry.

Results:

Mouse platelets perish rapidly when stored at RT, which is clearly shownin FIG. 30A. However, in the presence of mere 0.1 mM DANA in the storagemedia, approximately 5-fold more platelets were recovered (FIG. 30B).Further results for other concentrations of DANA are shown in FIG. 30Cand FIG. 30D.

Conclusion:

Sialidase inhibitor DANA is capable of effectively preserving mouseplatelets from deterioration, greatly improving the recovery andsurvival of the transfused platelets.

Example 9 Inhibition of the Proliferation and Biofilm Formation ofSerratia marcescens by Sialidase Inhibitor DANA

Bacterial contamination of blood products is currently the mostsignificant transfusion-associated infectious risk. Plateletconcentrates (PCs) are the most likely product to be contaminated due totheir storage conditions (22° C. with agitation, neutral pH, and highglucose content), which are particularly amenable to bacterial growth.Although Gram-positive bacteria are most commonly recovered fromcontaminated PCs, Gram-negative bacteria are more frequently associatedwith severe illness and fatality. Gram-negative Serratia marcescens is asignificant human opportunistic pathogen, which has been implicated innumerous adverse transfusion reactions (ATRs) involving contaminatedPCs. The ability of this species to survive under unfavourableenvironmental conditions, resist disinfection and formsurface-associated communities of micro-organisms (biofilms) presents achallenge for its elimination in the clinical environment. Recently, ithas been shown that the closely related species Serratia liquefaciensforms biofilms under platelet storage conditions, which is associatedwith reduced detection by colony counting.

In order to proliferate in platelet products, the contaminated bacteriaare likely to have a machinery to obtain and/or utilize sialic acid.Serratia marcescens is a Gram-negative bacterium that has beenimplicated in adverse transfusion reactions associated with contaminatedplatelet concentrates. It produces a range of extremely virulentproducts including proteases, nucleases, lipases, chitinases andhaemolysin; however, the presence of a secreatable sialidase has not yetbeen described. Based on the virulent characteristics of the secretedproducts by Serratia marcescens, the presence of sialidases is highlyplausible. Therefore, this strain was chosen to test oursialidase-inhibition strategy to inhibit bacterial growth. The Serratiamarcescens strain (ATCC #43862) has previously been used in studiesinvolving bacterial detection and growth in blood products.

Materials and Methods: Bacterial Strain and Growth Conditions:

Serratia marcescens strain (ATCC #43862) was purchased from AmericanType Culture Collection (Manassas, Va.). Cells were grown in brain-heartinfusion broth (ATCC media 3) at 37° C. and 250 rpm. Frozen stocks wereprepared from overnight culture and stored at −80° C. in brain-heartinfusion broth containing 15% glycerol by volume.

Biofilm Formation:

To prepare the seed culture, the cryostock of Serratia marcescens wasinoculated into 3 mL of brain-heart infusion broth with a cotton swaband incubated at 37° C. with agitation at 250 rpm for 6 h. The celldensity was determined at 600 nm on a dual wavelength spectrometer anddiluted to 0.5 McFarland Standard (1.5×10⁸ cells/mL) with sterile PBS.Ten μL of the diluted culture was inoculated into 140 μL of 30% plasmain PAS, 30% PC by volume in PAS or 100% plasma, supplemented with orwithout 1 mM DANA, in the wells of 96-well PVC plates (CorningBiosciences). For each media, six replicates were performed. Ten μL ofPBS was inoculated into the control wells. The microtiter plates werethen sealed with sterile porous film (VWR) and placed on a platformshaker. The cultures were incubated for 48 h with gentle shaking (˜100rpm). The cultures were gently mixed and transferred to a polystyreneplate for the determination of planktonic cell density at OD 595 nm. Thewells on the original microtiter plates were washed with 3×200 μL ofPBS, air dried, stained for 15 min with an aqueous solution of 0.1%(wt/vol) crystal violet, rinsed with water, and air dried for 1 hr. Thecrystal violet retained by the biofilm was eluted with 200 μL ofdimethyl sulfoxide (DMSO) or 30% acetic acid, and read at 595 nm.

Results:

Under suboptimal growth conditions on the microtiter plate, lacking ofadequate agitation and aeration, and low temperature, S. marcescens grewwell in pure plasma (FIG. 31A). The cell growths were dramaticallyretarded in 30% plasma or 30% PC in PPS. Remarkably, inclusion of 1 mMDANA in the growth media inhibited the bacterial growth under allconditions. In parallel with trends observed for bacterial growth, theformation of biofilm correlated well with planktonic cell density andnegatively impacted by the presence of DANA in the growth media (FIG.31B). The measurement of the A595 nm of the biofilm formation for thebacteria grown in plasma could not be accurately interpreted due to thesignal overflow, suggesting stronger biofilm formation in pure plasmathan in PPS-based media.

Conclusion:

Sialidase inhibitor DANA is capable of inhibiting the proliferation andbiofilm formation of S. marcescens when analyzed with 96-well PVC plate.The data also show that S. marcescens contains a previously unreportedmachinery to obtain and/or utilize sialic acid to proliferate and/orform biofilms.

Example 10 Variations in Platelet Surface Glycans Among HealthyVolunteers

Platelets have the shortest shelf-life of all major blood components andare the most difficult to store; these limitations complicate platelettransfusion practices. Dr. Slichter and colleagues (Puget Sound BloodCenter, Seattle, Wash.) have identified significant differences inrecovery and survival of transfused fresh radiolabeled autologousplatelets among healthy subjects. The cause of the inter-individualdifferences in platelet recovery and survival remains unclear. Wedemonstrated that the loss of sialic acid from the surfaces ofcold-stored and transfused platelets promotes their clearance by hepaticAsialoglycoprotein receptors (Ashwell Morell receptors). The loss ofplatelet surface sialic acid correlates with increases in surfacesialidase activity during platelet storage. Here we investigated whetherfresh platelets from individual donors exhibit differences in surfaceglycan exposure, which may affect post-transfusion platelet recovery andsurvival.

Material and Methods:

Venous blood was obtained from volunteers by venipuncture into 0.1volume of Aster Jandl citrate-based anticoagulant. Approval for blooddrawing was obtained from the Institutional Review Board of Brigham andWomen's Hospital, and informed consent was approved according to theDeclaration of Helsinki. Platelet-rich plasma (PRP) was prepared bycentrifugation at 125×g for 20 min and platelets were separated from theplasma proteins by gel-filtration through a small Sepharose 2B column.Isolated platelets were incubated for 20 min at room temperature with 10μg/mL of the β-galactose specific FITC-conjugated E. cristagalli lectin(ECL). The samples were diluted with 200 μL of PBS and immediatelyanalyzed by flow cytometry on a FACSCalibur flow cytometer (BecktonDickenson). The mean fluorescence intensity was determined in gatedplatelet population.

Results:

The presence of a terminal galactose on surface glycoproteins (i.e.glycans lacking of SA) on freshly-isolated platelets varies considerablyamong healthy subjects (three of five individuals had low levels ofexposed galactose (15.3±4.1, MFI), as expected. However, two subjectsexhibited considerably higher (2-7.5-fold) levels of galactose exposure.These results were confirmed using a second galactose-specific lectinRCA I, and by repeated measurements of the same individuals at twodifferent time points. Similarly, preliminary studies with plateletconcentrates demonstrated a remarkable variation in platelet surfacesialidase activity (FIG. 32), which correlated with rates of sialic lossduring platelet storage and possibly during platelet circulation invivo. Our results show that fresh platelets from healthy individualsvary in surface sialidase activity and sialic acid content.

These results indicate that the surface sialic acid could represent afactor that affects the recovery and survival of the transfused freshplatelets.

Example 11 General Procedure of Preparing Platelet Protection SolutionContaining a Sialidase Inhibitor, β-Galactosidase Inhibitor, or Both aSialidase Inhibitor and β-Galactosidase Inhibitor

The PPS of the present invention can be made as follows. The totalvolume of the bag is 500 mL.

To prepare a platelet protection solution, the following components ofUSP grade are obtained:

-   -   1) Electrolytes such as Na, Cl, K, Ca, and Mg.    -   2) An energy source such as glucose or citrate to sustain        aerobic metabolism.    -   3) A buffer such as phosphate.    -   4) Water for injection (WFI).    -   5) A sialidase inhibitor.

Table 2 provides the concentrations and amount (grams) of componentsincluding energy sources, buffers and electrolytes required to prepare1000 mL of platelet protection solution. Water is added in an amount of1000 mL and the solution is buffered to maintain a pH of pH 7.2.

Sialidase inhibitor such as DANA can be added from sterile 0.1-1000 mMstock solution in water to the desired concentrations. Similarly, aβ-galactosidase inhibitor can also be added from sterile 0.1-1000 mMstock solution in water to the desired concentrations.

TABLE 2 PPS 1b PPS 2b PPS 3b PPS 4b Component mM g/L mM g/L mM g/L mMg/L Dibasic sodium phosphate, anhydrous 7.15 1.015 7.15 1.015 7.15 1.0157.15 1.015 (Na₂HPO₄), USP Mono basic phosphate, monohydrate 2.24 0.3102.24 0.310 2.24 0.310 2.24 0.310 (NaH₂PO₄•H₂O), USP Sodium citrate,dihydrate 10.00 2.940 10.00 2.940 10.00 2.940 10.00 2.940(C6H5Na3O7•2H2O), USP Sodium acetate, trihydrate 29.98 4.080 29.98 4.08029.98 4.080 29.98 4.080 (CH₃COONa), USP Sodium chloride (NaCl), USP79.20 4.629 70.80 4.138 77.70 4.541 69.30 4.050 Potassium chloride(KCl), USP 5.00 0.373 5.00 0.373 5.00 0.373 5.00 0.373 Magnesiumchloride, hexahydrate 1.50 0.305 1.50 0.305 1.50 0.305 1.50 0.305(MgCl₂•6H₂O), USP Calcium chloride, dihydrate 0.00 0.000 0.00 0.000 1.000.147 1.00 0.147 (CaCl₂•2H₂O), USP Glucose (C6H12O6), USP 0.00 0.00016.80 3.028 0.00 0.000 16.80 3.028 DANA, sodium salt (solid or stock1.00 0.313 1.00 0.313 1.00 0.313 1.00 0.313 aqueous solution) Water forinjection, USP, to 1000 mL

TABLE 3 PPS 1c PPS 2c PPS 3c PPS 4c PPS 5 Component mM g/L mM g/L mM g/LmM g/L mM g/L Dibasic sodium phosphate, anhydrous 7.15 1.015 7.15 1.0157.15 1.015 7.15 1.015 7.2 1.017 (Na₂HPO₄), USP Mono basic phosphate,monohydrate 2.24 0.31 2.24 0.31 2.24 0.31 2.24 0.31 2.2 0.308(NaH₂PO₄•H₂O), USP Sodium citrate, dihydrate 10 2.94 10 2.94 10 2.94 102.94 10.8 3.176 (C6H5Na3O7•2H2O), USP Sodium acetate, trihydrate(CH₃COONa), 29.98 4.08 29.98 4.08 29.98 4.08 29.98 4.08 32.5 4.423 USPSodium chloride (NaCl), USP 79.2 4.629 70.8 4.138 77.7 4.541 69.3 4.0595.3 5.567 Potassium chloride (KCl), USP 5 0.373 5 0.373 5 0.373 5 0.3735.0 0.373 Magnesium chloride, hexahydrate 1.5 0.305 1.5 0.305 1.5 0.3051.5 0.305 1.5 0.305 (MgCl₂•6H₂O), USP Calcium chloride, dihydrate 0 0 00 1 0.147 1 0.147 0.0 0.0 (CaCl₂•2H₂O), USP Glucose (C6H12O6), USP 0 016.8 3.028 0 0 16.8 3.028 0.0 0.000 DANA, sodium salt (solid or stock 10.313 1 0.313 1 0.313 1 0.313 1.0 0.313 aqueous solution)1-Deoxygalactonojirimycin HCl (DGJ) 2 0.392 2 0.392 2 0.392 2 0.392 2.00.392 Water for injection, USP, to 1000 mL PPS 6 PPS7 PPS8 PPS9Component mM g/L mM g/L mM g/L mM g/L Dibasic sodium phosphate,anhydrous 7.2 1.017 7.2 1.017 7.2 1.017 7.2 1.015 (Na₂HPO₄), USP Monobasic phosphate, monohydrate 2.2 0.308 2.2 0.308 2.2 0.308 2.2 0.31(NaH₂PO₄•H₂O), USP Sodium citrate, dihydrate 10.8 3.176 10.8 3.176 10.83.176 10.0 2.94 (C6H5Na3O7•2H2O), USP Sodium acetate, trihydrate(CH₃COONa), 32.5 4.423 32.5 4.423 32.5 4.423 30.0 4.08 USP Sodiumchloride (NaCl), USP 86.8 5.071 95.3 5.567 86.8 5.071 70.8 4.138Potassium chloride (KCl), USP 5.0 0.373 5.0 0.373 5.0 0.373 5.0 0.373Magnesium chloride, hexahydrate 1.5 0.305 1.5 0.305 1.5 0.305 1.5 0.305(MgCl₂•6H₂O), USP Calcium chloride, dihydrate 0.0 0.0 0.0 0.0 0.0 0.00.0 0.0 (CaCl₂•2H₂O), USP Glucose (C6H12O6), USP 17.0 3.063 0.0 0.00017.0 3.063 16.8 3.028 DANA, sodium salt (solid or stock 1.0 0.313 0.00.000 0.0 0.000 0.0 0.0 aqueous solution) 1-Deoxygalactonojirimycin HCl(DGJ) 2.0 0.392 0.0 0.000 0.0 0.000 2.0 0.392 Water for injection, USP,to 1000 mLTable 3 shows various formulations when adding a β-galactosidaseinhibitor to the platelet protective solution. For example,PPS1a,c-PPS6a,c all have both a β-galactosidase inhibitor and asialidase inhibitor. PPS7 and PPS8 solutions do not include either;rather they are examples of solutions to which a β-galactosidase and/ora sialidase inhibitor can be added. PPS9 has a β-galactosidase inhibitorand not a sialidase inhibitor.

Example 12 Preservation of Mouse Platelets in PPS Containing a SialidaseInhibitor

Mouse platelets have a life span of approximately 4-5 days, considerablyshorter than human platelets (8-10 days). They are also much less stablethan human platelets when stored at room temperature or 4° C. However,these shortcomings of mouse platelets can be exploited to assess theefficiency of platelet protection solutions for the preservation ofplatelets.

Materials and Methods:

Mouse blood was obtained from anesthetized mice using 3.75 mg/g ofAvertin (Fluka Chemie, Steinheim, Germany) by retro-orbital eye bleedinginto 0.1 volume of Aster-Jandl anticoagulant and centrifuged at 200×gfor 8 min at RT. The supernatant, containing platelet rich plasma, buffycoat, and some RBC, was removed and centrifuged at 300×g for 6 min toobtain platelet rich plasma (PRP). Four 150 aliquots of PRP weretransferred to 4×1.5 mL Eppendorf tubes, and centrifuged at 1000×g for 5min. About 70% of the supernatant (105 μL) was removed from each tube,and replaced with equal volume of INTERSOL® solution. DANA and/orglucose (as a nutrient) were added to final concentrations of 1.0 and 10mM, respectively, from 100 mM stock solutions in PBS. The volumes intubes lacking one or both additives were evened out with PBS. Theplatelet suspensions were stored at RT for 48 h on a shaker and analyzedby flow cytometry.

Results:

Not surprisingly, mouse platelets deteriorated rapidly in INTERSOL®solution, when stored at RT (FIG. 33, panel Aa). Only 57% platelets weregated (FIG. 33, panel Ba). Remarkably, over 80% of the original plateletevents were counted within the platelet gate when stored with 1 mMsialidase inhibitor DANA (FIG. 33, panels Ab and Bb). Addition of 10 mMglucose resulted in even higher platelet counts recovery after storage(FIG. 33, panels Ac and Bc). A combination of both DANA and glucosepreserved all platelets (93% gated, FIG. 33, panels Ad and Bd). DANAalone or a combination with glucose results in a more resting plateletpopulation as judged by their forward and side scatter characteristics(the population is “less elongated”, i.e., formed less plateletaggregates) than glucose alone (FIG. 33, panels Ab and Ad, compare withFIG. 33, panel Ac). This data suggests that DANA is more effective thanglucose in preserving platelets in a resting state and in preservingplatelet numbers following platelet storage.

Conclusion:

Together, the data indicate that the presence of DANA during plateletstorage improves the quality of the stored platelets in at least 30%plasma in platelet additive INTERSOL® solution.

Example 13 Improved In Vitro Quality of Human Platelets Stored in Plasmain the Presence of Sialidase Inhibitor DANA

The state of a “healthy” platelet is partially defined by its shape andsize. Platelet shape change and aggregation are hallmarks of plateletactivation. Once activated, platelets change shape and secrete theirgranular contents. Storage of platelets is accompanied by plateletactivation, i.e. platelet shape change and granule release. Humanplatelets also increase surface sialidase expression and lose surfacesialic acid during storage. Presumably, sialidases are stored ingranules and are released to the platelet surface during storage. Theresults from Example 12 suggest that mouse platelets may also losesialic acid during storage and this process can be effectively inhibitedby the presence of sialidase inhibitor DANA in the storage, greatlyimproving the post-transfusion recovery and survival of platelets. Thedata further indicate that the quality of stored human platelets can beimproved by including a sialidase inhibitor in the storage media.

Resting platelets have a discoid shape and produce differentside-scatter (SSC) signals in the flow cytometer, depending on theirrelative orientation to the laser beam. A resting platelet populationhas a wide (“round”) distribution in the SSC/FSC signal. Uponstimulation, platelets form pseudopods and become spherical (shapechange) thereby producing a characteristic SSC signal irrespective oftheir relative orientation to the laser beam. Therefore, an activatedplatelet population appears more “condensed” on a FCS/SSC plot.

Based on these considerations, we investigated if DANA affects humanplatelet activation (i.e. shape change and granule release) duringstorage in plasma.

Materials and Methods:

Venous blood was obtained from volunteers by venipuncture into 0.1volume of Aster Jandl citrate-based anticoagulant. Approval for blooddrawing was obtained from the Institutional Review Board of Brigham andWomen's Hospital, and informed consent was approved according to theDeclaration of Helsinki. Platelet-rich plasma (PRP) was prepared bycentrifugation at 125×g for 20 min and platelets were separated from PRPafter adding PGE1 (1 μg/mL) by centrifugation for 5 min at 850×g. Thesupernatant (platelet-poor plasma, PPP) was saved. The platelet pelletwas resuspended in PPP, ½ volumes of original PRP, and divided intoaliquots. DANA was added to 1.0 mM from 100 mM stock in PBS to half ofthe aliquots, only PBS was added to the controls. The samples werestored in the wells of a 96-well microtiter plate covered with agas-permeable film with agitation on a shaker at room temperature.Platelet size and density were measured by forward (FSC) and sidescatter (SSC) on a FACSCalibur flow cytometer (BD). Platelets were gatedby their forward and side scatter characteristics. For the analysisplatelet degranulation, i.e., a-granule release, stored platelets wereanalyzed for P-selectin surface expression by incubating with 0.1 μg/mLof FTIC mouse anti-human CD62P (BD Pharmingen) antibody in 50 μL of PBSfor 30 min at RT. The mixture was then diluted with 200 μL of PBS andimmediately analyzed by flow cytometry. The percentage of P-selectin(FITC)-positive cells was determined in gated platelet population.

Results:

After 72 h storage at RT in plasma, human platelets displayed a decreasein side and forward scatter characteristics (FIG. 34, panel A, leftside) compared with fresh RT platelets (not shown). The decrease in sideand forward scatter characteristics is characteristic for plateletactivation. In contrast, addition of 0.5 mM DANA during platelet storageled a visible improvement of the platelet shape (FIG. 34, panel B, rightside). Comparisons of histograms of platelet count/SSC showed thatplatelets stored with DANA have increased mean fluorescence intensity(MFI), (FIG. 34, panel B, left side, note that the profile migratesslightly to the right side), suggesting that platelets stored in thepresence of DANA have higher granularity or internal complexity and areless activated. Similarly, histograms of platelet count/FSC histogramsshowed that platelets stored with DANA have higher side scatter meanfluorescence intensity (FIG. 34, panel B, right side, note that theprofile migrates slightly to the right side). These results show thatplatelets stored in the presence of DANA are bigger and retain adiscoid, resting shape.

These results were confirmed by analyzing the P-selectin exposure of thestored platelets with FTIC mouse anti-human CD62P (P-selectin) antibody(FIG. 35). Inclusion of DANA during storage significantly prevented theexposure of P-selectin, and inhibited α-granule release.

Together, the data indicate that the presence of DANA during plateletstorage improves the quality of the stored platelets in 100% plasma.

Example 14 Improved In Vitro Quality of Human Platelets Stored in PPSsContaining Sialidase Inhibitor DANA

Data described in Example 12 demonstrated that sialidase inhibitor DANAcan effectively preserve the quality of mouse platelets stored 30%plasma in platelet additive solution referred to as INTERSOL® solution.Data described in Example 13 clearly showed that DANA is also effectivefor preserving the quality of human platelets in 100% plasma. In thisExample, the studies were extended to human platelets stored inplasma/PAS in a ratio of 30:70, in the absence or presence of DANA.

Materials and Methods:

Human platelets were obtained as described in Example 13. The plateletpellet was resuspended in PPP, ⅕ volumes of original PRP, and aliquotedinto wells of a 96-well microtiter plate (60 μL per well). PAS(designated as PASa), containing 7.15 mM Na₂HPO₄, 2.24 mM NaH₂PO4, 10 mMsodium citrate, 30 mM sodium acetate, 79.2 mM NaCl, 5.0 mM KCl, and 1.5mM MgCl₂, was added to corresponding wells at 140 μL per well, DANA wasadded to 0, 0.1, and 0.5 mM from 10 or 100 mM stock in PBS to properwells. The sample volumes in the wells were evened out with PBS. Theplate was then covered with a gas-permeable film and placed on a shaker.Platelet size and density were measured by forward (FSC) and sidescatter (SSC) on a FACSCalibur flow cytometer (BD) at Day 7, and pH waschecked at Day 9.

Results:

All storage samples maintained at pH 6.8 after 9 days, demonstratingthis PPS formulation has enough buffer capacity for storing plateletsfor at least 9 days. In contrast, under similar storage conditions in100% plasma, the pH of the stored platelet samples dropped below pH 6.5.Significant deterioration of human platelets was noted after 7 days ofstorage at RT when stored in 30% plasma and 70% PPS solution. As shownin FIG. 36, panel A, only 55% of the total acquired events were gated inthe gate defined for fresh platelets (G1) while more than 40% of theacquired total events were platelet microparticles (plateletmicroparticles are considered as a readout of platelet deterioration)defined in G2. In contrast, when platelets were stored in the presenceof 0.1 mM DANA over 70% of the total acquired events were gated asplatelets (FIG. 36, panel B, G1). Accordingly, a dramatic reduction ofmicroparticle formation from 41.5% (FIG. 36, panel A, G2) to 23.95%(FIG. 36, panel B, G2) was observed. Increase of DANA concentration inthe storage media to 0.5 mM further increased platelet counts (81.6%gated, FIG. 36, panel C, G1) and reduced the formation of microparticles(13.52%, G2). Of particular note is that the platelet population appears“resting” upon addition of DANA to the storage solution, as judged bytheir side and forward scatter characteristics.

Conclusion:

Consistent with results described in Examples 12 and 13, DANA caneffectively preserve the quality of human platelets in 30% plasma in aplatelet protection solution, i.e., reduce platelet activation andmicroparticle formation, showing that a sialidase inhibitor such as DANAcan be used as an important component in PPS formulations for plateletstorage.

Example 15 Variability of Platelet Surface Sialidase Activities AmongHealthy Individuals and Up-Regulation of these Activities DuringPlatelet Storage at RT

Platelets have the shortest shelf life of all major blood components andare the most difficult to store; these limitations complicate platelettransfusion practices. The loss of sialic acid from the surfaces ofcold-stored and transfused platelets promotes clearance of platelets byhepatic Asialoglycoprotein receptors (Ashwell-Morell receptors). Theloss of platelet surface sialic acid correlates with increases insurface sialidase activity during platelet storage under refrigeration.Significant differences have been identified in recovery and survival oftransfused fresh radiolabeled autologous platelets among healthysubjects. The cause of the inter-individual differences in plateletrecovery and survival remains unclear. Here, we investigated whetherfresh platelets from individual donors exhibit differences in surfacesialidase expression that may lead to differential β-galactose exposureand affect post-transfusion platelet recovery and survival.

Methods:

Platelets were isolated from platelet concentrates (PC) stored underblood banking conditions by centrifugation, washed, re-suspended at aconcentration of 1-10×10⁹/mL in 140 mM NaCl, 3 mM KCl, 0.5 mM MgCl₂, 5mM NaHCO₃, 10 mM glucose, and 10 mM Hepes, pH 7.4 (buffer A). Plateletsialidase activity was determined by incubation of platelets (˜10⁸platelets) at 37° C. with 125 μM2′-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (4-MU-NANA) in 100mM NaOAc (pH 5.0), 80 mM NaCl. Reaction mixtures were sampled at varioustime points and the reactions were quenched with 1.5 volumes of 200 mMglycine/NaOH (pH 10.4). After clarification by centrifugation, thesamples were read on a fluorescence plate reader with 355 nm excitationat 460 nm emission.

Results:

Each donor has readily detectable sialidase activity after 1 day storageat RT, which was up-regulated after prolonged storage (Day 6). Plateletsfrom Donor B exhibited higher sialidase activity on both Day 1 and Day6. See FIG. 42.

Conclusion:

The difference of platelet surface sialidase expression among individualdonors suggests a possible difference of platelet surface β-galactoseexposure among individual donors.

Example 16 Variability of Platelet Surface β-Galactosidase ActivitiesAmong Healthy Individuals and Up-Regulation of these Activities DuringPlatelet Storage at RT

Mammalian neuraminidases have been classified as lysosomal (Neu1),cytosolic (Neu2), plasma membrane (Neu3), and mitochondria/lysosomal(Neu4) based on their subcellular distributions, pH optima, kineticproperties, responses to ions and detergents, and substratespecificities. Of the four sialidases, only Neu1 is ubiquitouslyexpressed at different levels in various tissues and cell types. Theimportance of these proteins in normal cellular physiology isillustrated by the numerous metabolic processes that they control,including cell proliferation and differentiation, cell adhesion,membrane fusion and fluidity, immunocyte function and receptormodification.

Neu1 initiates the intralysosomal hydrolysis of sialo-oligosaccharides,-glycolipids, and -glycoproteins by removing their terminal sialic acidresidues. In human and murine tissues, Neu1 forms a complex with atleast two other proteins, β-galactosidase and the protectiveprotein/cathepsin A (PPCA). By virtue of their association with PPCA,Neu1 and β-galactosidase acquire their active and stable conformation inlysosomes. However, PPCA appears to function as a crucialchaperone/transport protein for Neu1. Because Neu1 is poorly mannose6-phosphorylated, it depends on PPCA for correct compartmentalizationand catalytic activation in lysosomes. Only a small amount of PPCA andβ-galactosidase activities is found in the Neu1-PPCA-β-galactosidasecomplex, which instead contains all of the Neu1 catalytic activity. Byunderstanding how and when Neu1 and PPCA interact, how they regulateeach other in different cell types, and what determinants control theirassociation, important insight is gained regarding their significance inphysiologic and pathologic conditions.

As described herein, we have previously demonstrated that Neu1 isrearranged to platelet surface after platelet refrigeration. Since theassociation with β-galactosidase goes along with Neu1 activity, thesurface expression and up-regulation (during storage at RT) of Neu1activity suggests similar observations for β-galactosidase activity. Totest this hypothesis, we analyzed the platelet β-galactosidase activitybefore and after platelet storage.

Methods:

Platelets were isolated from platelet concentrates stored under bloodbanking conditions by centrifugation, washed, re-suspended in plateletwash Buffer A and counted by flow cytometry. Platelet β-galactosidaseactivity was determined by incubation of washed platelets (˜5-10⁸platelets) or PC at 37° C. with 2.5 mM Galβ-pNP in 100 mM NaOAc (pH5.0), 80 mM NaCl. Reaction mixtures were sampled at various time pointsand the reactions were quenched with 1.5 volumes of 200 mM glycine/NaOH(pH 10.4), clarified by centrifugation and read on a spectrophotometerplate reader at 405 nm.

Results:

β-Galactosidase activity was readily detected with washed platelets ordirectly with platelet concentrates. Enzyme activity varies amongdonors, but is up-regulated during platelet storage. It is noted thatDonor B, exhibiting higher sialidase activity, also exhibited higherβ-galactosidase activity. See FIG. 43.

Example 17 Isolated Platelets from Healthy Volunteers Differ in Terminalβ-Galactose Content, and this Correlates with Platelet Ingestion byHepG2 Cells In Vitro

Platelet surface sialidase catalyzes the release of sialic acids fromthe platelet surface and exposes β-galactose residues. The presence ofβ-galactosidase on the platelet surface suggests that the plateletsurface β-galactose exposure may vary among individuals and over thecourse of platelet storage. To test this hypothesis, we obtainedplatelets from healthy volunteers from platelet concentrates andmeasured for β-galactose exposure by RCA lectin binding.

We have shown that the hepatoma cell line HepG2 ingestssialyltransferase-deficient mouse platelets (ST3Gal-IV^(−/−) platelets)and sialic acid-deficient, refrigerated human platelets in vitro. Thiscell line expresses the Asgr (Ashwell Morell Receptor), whichspecifically recognizes platelets in vitro and in vivo. See FIG. 37.Whether platelets with high or low terminal β-galactose initiateendocytosis by hepatocytes will be determined in HepG2 cultures.

Methods:

Platelets are isolated by centrifugation, washed with PBS, andresuspended in PBS, ⅕ of original plasma volume. Platelets are countedby flow cytometry and then diluted appropriately. Lectins are dilutedappropriately in PBS. Five μl of diluted platelets is added to the 100μL of lectin and incubated for 15 min. After incubation, 300 μl PBS isadded to lectin-platelet solution and analyzed by a flow cytometer.

For the HepG2 assay, isolated human platelets were labeled withCM-Orange, added to HepG2 cells and incubated for 30 min at 37° C. Thenumber of platelets in the media was counted by flow cytometry. Thenumber of platelets added to HepG2 cells was set to 100% for eachindividual. Ingestion of fluorescently (CM-orange) labeled freshplatelets was detected using flow cytometry as an increase in hepatocyteassociated orange fluorescence.

Results:

The presence of a terminal β-galactose on surface glycoproteins (e.g.,glycans lacking sialic acid) on freshly-isolated platelets variesconsiderably among healthy subjects as measured by RCA-I lectin bindingassay (FIG. 38). Platelets from subject 1 have the highest surfaceβ-galactose exposure, while those from subject 6 have the lowest surfaceβ-galactose exposure. These findings were confirmed by HepG2 assay. SeeFIGS. 39A and 39B.

Conclusion:

Our results show that fresh platelets have variable surface β-galactoseexposure/sialic acid loss among healthy individuals.

Example 18 Terminal β-Galactose Content Decreases on Platelet SurfacesOver the Course of Platelet Storage and Correlates with Ingestion byHepG2 Cells

In this Example, we extended our studies as described in Example 17 toplatelets isolated from platelet concentrates stored under standardblood banking conditions.

Results:

During storage at RT, platelet surface β-galactose exposure appears topeak at day 2, then decrease during further storage (See FIG. 40), whichwas confirmed by HepG2 assay. See FIGS. 41A and 41B

Summary:

Human platelets have variable (among donors) surface sialidase andβ-galactosidase activities, both of which are up-regulated duringplatelet storage at RT. In addition, human platelets have variablesurface β-galactose exposure/sialic acid loss among individual donors.During storage at RT, platelet surface β-galactose exposure appears topeak at day 2, then decrease during further storage. Since theassociation with β-galactosidase goes along with Neu1 sialidaseactivity, the concerted up-regulation of sialidase and β-galactosidaseactivities on platelet surface indicates that the multi-enzyme complexis relocated from lysosome to platelet surface during plateletstorage/aging, possibly through the fusion between platelet membrane andlysosomal membrane (FIG. 37). The relocation of both Neu1 andβ-galactosidase onto platelet surface catalyzes the sequentialdegradation of platelet surface glycans, loss of sialic acid, followedby β-galactose, exposing terminal N-acetylglucosamine (GlcNAc). GlcNAccan potentially be further removed, exposing the mannose residues.Additionally, the mannose residues can be readily recognized bymacrophage mannose receptors, triggering immediate platelet clearance.

Example 19 Fresh Platelets Bear Terminal β-Galactose, which is ReadilyCleaved by β-Galactosidase Exposing β-GlcNAc Thereby Leading toIngestion by THP-1 Cells

We treated fresh isolated platelets from healthy volunteers withβ-galactosidase, which cleaves terminal β-galactose from plateletsurfaces.

Results:

Fresh platelets treated with β-galactosidase are readily ingested(4-fold increase) by the macrophage-like cell line THP-1 when comparedto control platelets. These results show that fresh platelets haveterminal β-galactose, which can be readily accessed and cleaved byβ-galactosidase. This maneuver exposes underlying (-GlcNAc residues.Exposure of β-GlcNAc presumably promotes ingestion of platelets via theαMβ2 macrophage receptor. See FIG. 44.

Summary:

Fresh isolated platelets have exposed β-galactose showing that plateletscontain desialylated glycans. Removal of β-galactose usingβ-galactosidase exposes terminal N-acetylglucosamine (GlcNAc), andexposure of GlcNAc leads to ingestion of platelets by THP-1 cells, andby macrophages. GlcNAc can potentially be further removed, exposing themannose residues. Furthermore, the mannose residues can be readilyrecognized by macrophage mannose receptors, triggering immediateplatelet clearance.

Example 20 Improved In Vitro Quality of Human Platelets Stored in V-PAS™Solution Containing β-Galactosidase Inhibitor DGJ

Data described in Example 19 demonstrated that loss of β-galactose fromplatelet surface leads to increased ingestion of platelets byα_(M)β₂-expressing THP-1 cells. Whether inhibition of β-galactose lossfrom platelet surface during platelet storage may improve the thequality of stored platelets was tested. Platelets were stored inplasma/V-PAS™ solution in a ratio of 30:70, in the absence or presenceof β-galactosidase inhibitor DGJ (1-deoxygalactonojirimycin), andanalyzed the stored platelets over the course of storage. V-PAS™solution is used herein to refer to a platelet protection solutionhaving a silaidase inhibitor, and one or more storage medium components(e.g., not having a β-galactosidase inhibitor). V-PAS+™ solution orV-PAS+2 are used herein to refer to a platelet protection solutionhaving a silaidase inhibitor, a β-galactosidase inhibitor, and one ormore storage medium components. In some of the figures and examples, theterm V-PAS, V-PAS+ or V-PAS+2 can be shown with or without the “-” asVPAS, VPAS+, VPAS+2, respectively.

Materials and Methods:

ABO-matched random donor platelet concentrates (Blood TransfusionService, Massachusetts General Hospital) were pooled, aliquoted into50-mL conical tubes and centrifuged (1000×g, 20 min). After removal of70% plasma, the pelleted platelets were allowed to rest for 1 hour,re-suspended in the remaining plasma, and pooled to homogenize theplatelet suspension. The resultant platelet suspension was divided intoPermaLife bags (PL 30, OriGen Biomedical) (7.2 mL/bag), to which 16.8 mLof plasma or V-PAS™ or V-PAS+(a combination of V-PAS with 2 mM DGJ) wasadded per bag. The platelet bags were placed on a platelet rotator andstored at room temperature. The platelet aliquots were sampled on Day 1,Day 5, Day 7, or Day 9 and diluted with PBS. The diluted platelets werestained with FITC-labeled Annexin V for PS exposure, or FITC-labeledCD62P antibodies for P-selectin exposure, and analyzed by flowcytometry.

Results:

Phosphatidylserine (abbreviated PS) is a phospholipid component, usuallykept on the inner-leaflet (the cytosolic side) of cell membranes by anenzyme called flippase. When a cell undergoes apoptosisphosphatidylserine is no longer restricted to the cytosolic part of themembrane, but becomes exposed on the surface of the cell. Fresh isolatedplatelets have little, but readily detectable, surface exposure of PS,which can be measured by Annexin V binding. Upon storage, PS exposure onplatelet surface is increased. Increased surface exposure of PS onstored platelets has been correlated with reduced platelet recoveryafter transfusion. The platelet PS surface exposure during plateletstorage were monitored under different conditions at the indicated timepoints in FIG. 45 and FIG. 46.

As shown in FIG. 45A, platelets stored in 100% plasma (Plasma Platelet)demonstrated a continuous increase in PS exposure, as measured byAnnexin V binding, which is (roughly) linearly proportional to thestorage time. As expected, platelets stored in both plasma and V-PAS(V-PAS Platelet) also demonstrated gradual increase of PS exposure overthe course of the 7-day storage, but at much slower pace as compared toplasma alone. However, PS exposure on V-PAS Platelets increased afterDay 5 although it is still much lower than that found on PlasmaPlatelets at Day 7. Introduction of DGJ to V-PAS solution (i.e., V-PAS+solution) shows a similar impact on platelet surface exposure of PS upto Day 5 as compared to V-PAS platelets (i.e., without DGJ). However,after Day 5, V-PAS+ inhibits accelerated PS exposure, as compared tothat seen in platelets stored in the presence of V-PAS (SeeV-PAS+Platelet in FIG. 45B). Consistently, the difference of PS exposurebetween V-PAS Platelets and V-PAS+Platelets at Day 7 is significant(FIG. 45B). Both V-PAS platelets and V-PAS+ platelets are significantlybetter than plasma platelets. These data strongly suggest that DGJimproves the quality of platelets subjected to prolonged storage. Toconfirm this preliminary but important observation, paired studies wereperformed between Plasma Platelets and V-PAS+Platelets, which werestored beyond 7 days. Data from Plasma Platelets confirmed the linearrelationship between platelet surface PS exposure and storage time asobserved previously for platelets stored for 7 days, and suchrelationship can be extended to 9 days (FIG. 46A). A similar observationwas made for V-PAS+Platelets. However, the increase in PS exposure onV-PAS+Platelets (slope=0.9402±0.1062) is much slower than plasmaplatelets (slope=1.765±0.2111), suggesting that V-PAS+ is a betterstorage medium than plasma.

P-selectin expression (i.e., platelet granule secretion) on plateletsurface is used to evaluate the quality of stored platelets. Itsexpression on platelet surface is independent of PS exposure. Thedramatic down-regulation of PS exposure on V-PAS+Platelets compared toPlasma Platelets led us to examine how V-PAS+ impacts the plateletactivation after storage for 9 days. As shown in FIG. 46B, V-PAS+ hassignificant negative effect on the P-selectin exposure on plateletsstored for 9 days compared with plasma, indicating that platelets storedin V-PAS+ have less platelet activation than those stored in 100%plasma.

Conclusion:

DGJ can effectively preserve the quality of human platelets, i.e.,reduce platelet apoptosis, and platelet activation, when stored in 30%plasma and in the presence of a platelet protective solution.Collectively, our data show that a β-galactosidase inhibitor such as DGJcan be used as an effective component in PPS formulations for plateletstorage.

Example 21 Survival of Platelets after Transfusion Platelet Counts andPreparation:

Blood was obtained from anesthetized 8 weeks old C57/Bl6 mice byretroorbital bleeding. Platelets and platelet poor plasma (PPP) wereprepared by differential centrifugation as described (Hoffmeister, K.M., et. al., “The clearance mechanism of chilled blood platelets”, Cell112:87 (2003)). Platelets were stored in 100% plasma, or in a mixture of30% plasma and 70% VPAS, and 30% plasma and 70% VPAS+2. VPAS+2 issometimes also referred to as V-PAS+ herein, and includes aβ-galactosidase inhibitor as well as a sialidase inhibitor. In thisembodiment, V-PAS+ includes DGJ as the β-galactosidase inhibitor andDANA as the sialidase inhibitor. Platelet numbers were adjusted prior totransfusion to ensure equal numbers of transfused platelets percondition. Fresh platelets were kept in 100% plasma and transfusedimmediately after isolation.

Platelet Transfusions:

Isolated platelets were labeled with 2.5 μM of CMFDA for 15 min.Following staining, platelets were pelleted and resuspended in 500 μl ofplasma, V-PAS/Plasma (70:30) or V-PAS+/Plasma (70:30). Platelets werestored for 20 hours at room temperature and transfused into 8 weeks oldsyngeneic mice. Non-stored fresh platelets in plasma were used ascontrol. Platelet survivals were determined by intravenous injectionsCMFDA-labeled mouse platelets, as described in Rumjantseva V., et al.,“Dual roles for hepatic lectin receptors in the clearance of chilledplatelets”, Nature Medicine 15(11): 1273-80 (2009) and Sorensen A. L.,et al., “Role of sialic acid for platelet life span: exposure ofbeta-galactose results in the rapid clearance of platelets from thecirculation by asialoglycoprotein receptor-expressing liver macrophagesand hepatocytes”, Blood 114(8):1645-54 (2009). Blood was collected byretroorbital bleeding at 5 min, 2 hours, and 48 hours. The percentagesof CMFDA-positive platelets were determined by flow cytometry. Theamount of fluorescent platelets at 5 minutes is shown in FIG. 47.Short-term (2 hours) and long-term (48 hours) survivals of thetransfused platelet populations are shown in FIG. 48.

Example 22 Preservation of Mouse Platelets in PAS Containing aGalactosidase Inhibitor DGJ

Mouse platelets have a life span of approximately 4-5 days, considerablyshorter than human platelets (8-10 days). They are also much less stablethan human platelets when stored at room temperature or 4° C. However,these shortcomings of mouse platelets can be exploited to assess theefficiency of platelet additive solutions for the preservation ofplatelets.

Materials and Methods: Blood was obtained from anesthetized 8 weeks oldC57/Bl6 mice by retroorbital bleeding. Platelets and platelet poorplasma (PPP) were prepared by differential centrifugation as described.Isolated platelets were labeled with 2.5 μM of CMFDA for 15 min.Following staining, platelets were pelleted and resuspended in 500 μl ofplasma, or in a mixture of 30% plasma and 70% pPAS (i.e., PPS9formulation in Tables 1 and 3 without the 2 mM of DGJ shown in thetables) or pPAS+DGJ (2 mM) (PPS9 formulation in Tables 1 and 3).Platelets were stored for 20 hrs at room temperature and transfused into8 weeks old syngeneic mice. Non-stored fresh platelets in plasma wereused as control. Platelet survivals were determined by intravenousinjections CMFDA-labeled mouse platelets, respectively. Blood wascollected by retroorbital bleeding at 5 min, 2, 24, 48 and 72 hrs. Thepercentages of CMFDA-positive platelets were determined by flowcytometry. The survivals of the transfused platelet populations areshown in FIG. 49.

Result: Mouse platelets survived poorly in vivo following 20-hr storageat RT in 100% plasma and even worse a 30%:70% mixture of plasma andpPAS. However, addition of 2 mM DGJ in pPAS during platelet storagesignificantly improved the survival of the transfused platelets.

Result: Together, the data indicate that the presence of DGJ duringplatelet storage improves the quality of the stored platelets in 30%plasma in platelet additive solution.

This application relates to U.S. application Ser. No. 13/474,473,entitled “Increased In Vivo Circulation Time Of Platelets After StorageWith a Sialidase Inhibitor” filed May 17, 2012, by Karin Hoffmeister,Qiyong Peter Liu, and Robert Sackstein; U.S. application Ser. No.13/474,627, entitled “Improved Platelet Storage and Reduced BacterialProliferation in Platelet Products Using a Sialidase Inhibitor” byQiyong Peter Liu and Karin Hoffmeister; U.S. application Ser. No.13/474,679, entitled “Platelet Additive Solution Having a SialidaseInhibitor” filed May 17, 2012, by Qiyong Peter Liu and KarinHoffmeister; and PCT Application No. PCT/US2012/038462 entitled“Improved Platelet Storage Using a Sialidase Inhibitor” by Qiyong PeterLiu, Karin Hoffmeister and Robert Sackstein. This application alsorelates to U.S. Provisional Application No. 61/613,876, filed Mar. 21,2012; U.S. Provisional Application No. 61/613,837, filed Mar. 21, 2012;U.S. Provisional Application No. 61/503,984, filed Jul. 1, 2011; U.S.Provisional Application No. 61/487,077, filed May 17, 2011; U.S.Provisional Application No. 61/710,273, filed Oct. 5, 2012; U.S.Provisional Application No. 61/814,325, filed Apr. 21, 2013; and U.S.Provisional Application No. 61/813,885, filed Apr. 19, 2013. Therelevant teachings of all the references, patents and/or patentapplications cited herein are incorporated herein by reference in theirentirety.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A platelet protection solution (PPS), comprising:a. an amount of one or more β-galactosidase inhibitors and an amount ofone or more sialidase inhibitors, and optionally an amount of one ormore glycan-modifying agents; b. one or more of PPS components thatincludes a salt, a citrate source, a carbon source, or any combinationthereof, wherein the one or more PPS components comprises: a sodiumsource in an amount ranging between about 100 mM and about 300 mM; achloride source in an amount ranging between about 40 mM and about 110mM; a citrate source in an amount ranging between about 2 mM and about20 mM; an acetate source in an amount ranging between about 10 mM andabout 50 mM; a phosphate source in an amount ranging between about 5 mMand about 50 mM; a potassium source in an amount ranging between about0.5 mM and about 10 mM; and a magnesium source in an amount rangingbetween about 0.5 mM and about 5.0 mM.
 2. The PPS of claim 1, furthercomprising at least one of the group consisting of: a. a calcium sourcein an amount ranging between about 0.1 mM and about 2.5 mM; b. a glucosesource in an amount ranging between about 0.1 mM and about 30 mM; and c.both.
 3. The PPS of claim 1, wherein the chloride source is present inthe amount ranging between about 90 mM and about 110 mM;
 4. The PPS ofclaim 1, wherein the PPS is maintained at a pH ranging between about 6.4and about 7.6.
 5. The PPS of claim 1, wherein the phosphate source isselected from the group consisting of sodium monophosphate, sodiumdiphosphate, sodium triphosphate, and a combination thereof.
 6. The PPSof claim 1, wherein the citrate source is selected from the groupconsisting of monosodium citrate, disodium citrate, trisodium citrate,citric acid, and a combination thereof.
 7. The PPS of claim 1, whereinthe the acetate source is selected from the group consisting of sodiumacetate, potassium acetate, magnesium acetate, and a combinationthereof.
 8. The PPS of claim 1, wherein the sodium source is selectedfrom the group consisting of sodium chloride, sodium citrate, sodiumacetate, sodium phosphate, and a combination thereof.
 9. The PPS ofclaim 1, wherein the chloride source is selected from the groupconsisting of sodium chloride, magnesium chloride, potassium chloride,and a combination thereof.
 10. The PPS of claim 1, wherein the potassiumsource is selected from the group consisting of potassium chloride,potassium citrate, potassium acetate, potassium phosphate, potassiumsulfate, and a combination thereof.
 11. The PPS of claim 1, wherein themagnesium source is selected from the group consisting of magnesiumchloride, magnesium citrate, magnesium sulfate, and a combinationthereof.
 12. The PPS of claim 9, wherein the calcium source is selectedfrom the group consisting calcium chloride, calcium acetate, calciumcitrate, and a combination thereof.
 13. The PPS of claim 1, wherein thesialidase inhibitor is selected from the group consisting of: fetuin;2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA);ethyl(3R,4R,5S)-5-amino-4-acetamido-3-(pentan-3-yloxy)-cyclohex-1-ene-1-carboxylate);(2R,3R,4S)-4-guanidino-3-(prop-1-en-2-ylamino)-2-((1R,2R)-1,2,3-trihydroxypropyl)-3,4-dihydro-2H-pyran-6-carboxylicacid;(4S,5R,6R)-5-acetamido-4-carbamimidamido-6-[(1R,2R)-3-hydroxy-2-methoxypropyl]-5,6-dihydro-4H-pyran-2-carboxylicacid;(1S,2S,3S,4R)-3-[(1S)-1-acetamido-2-ethyl-butyl]-4-(diaminomethylideneamino)-2-hydroxy-cyclopentane-1-carboxylicacid; a combination thereof; and a pharmaceutically acceptable saltthereof.
 14. The PPS of claim 1, wherein the one or more β-galactosidaseinhibitors are selected from the group consisting of:1-deoxygalactonojirimycin (DGJ); N-(n-butyl)deoxygalactonojirimycin;N-(n-nonyl)deoxygalactonojirimycin; 5-deoxy-L-arabinose; galactostatinbisulfate; 3′,4′,7-trihydroxyisoflavone; D-ribonolactone;N-octyl-4-epi-β-valienamine; phenylethyl β-D-thiogalactopyranoside;difluorotetrahydropyridothiazinone; 4-aminobenzyl1-thio-β-D-galactopryranoside; a combination thereof; and apharmaceutically acceptable salt thereof.
 15. A platelet compositioncomprising: a. isolated platelets; b. a PPS that comprises: i. one ormore β-galactosidase inhibitors and one or more sialidase inhibitors,and optionally one or more glycan-modifying agents; and ii. one or moreof PPS components that includes a salt, a citrate source, a carbonsource, or any combination thereof; and c. plasma; wherein the plateletcomposition is maintained at a pH ranging between about 6.4 and about7.6; and wherein the one or more PPS components comprises: a sodiumsource in an amount ranging between about 100 mM and about 300 mM; achloride source in an amount ranging between about 40 mM and about 110mM; a citrate source in an amount ranging between about 2 mM and about20 mM; an acetate source in an amount ranging between about 10 mM andabout 50 mM; a phosphate source in an amount ranging between about 5 mMand about 50 mM; a potassium source in an amount ranging between about0.5 mM and about 10 mM; and a magnesium source in an amount rangingbetween about 0.5 mM and about 5.0 mM.
 16. The platelet composition ofclaim 15, further comprising at least one of the group consisting of: a.a calcium source in an amount ranging between about 0.1 mM and about 2.5mM; b. a glucose source in an amount ranging between about 0.1 mM andabout 30 mM; and c. both.
 17. The platelet composition of claim 15,wherein the chloride source is present in the amount ranging betweenabout 90 mM and about 110 mM.
 18. The platelet composition of claim 15,wherein the plasma is present in an amount ranging between about 1% andabout 50% by volume.
 19. The platelet composition of claim 15, whereinthe platelet protection solution is present in an amount ranging betweenabout 50% and about 99% by volume.
 20. A bag or container thatcomprises: a. a bag or container suitable for platelet storage; and b. aPPS comprising: i. an amount of one or more β-galactosidase inhibitorsand an amount of one or more sialidase inhibitors, and optionally anamount of one or more glycan-modifying agents, or a combination thereof;and ii. one or more of PPS components that includes a salt, a citratesource, a carbon source, or any combination thereof wherein the one ormore PPS components comprises: a sodium source in an amount rangingbetween about 100 mM and about 300 mM; a chloride source in an amountranging between about 40 mM and about 110 mM; a citrate source in anamount ranging between about 2 mM and about 20 mM; an acetate source inan amount ranging between about 10 mM and about 50 mM; a phosphatesource in an amount ranging between about 5 mM and about 50 mM; apotassium source in an amount ranging between about 0.5 mM and about 10mM; and a magnesium source in an amount ranging between about 0.5 mM andabout 5.0 mM.
 21. The bag or container of claim 20, further comprisingat least one of the group consisting of: a. a calcium source in anamount ranging between about 0.1 mM and about 2.5 mM; b. a glucosesource in an amount ranging between about 0.1 mM and about 30 mM; and c.both.
 22. The bag or container of claim 20, wherein the chloride sourceis present in the amount ranging between about 90 mM and about 110 mM.23. The bag or container of claim 20, further comprising isolatedplatelets.
 24. The bag or container of claim 20, wherein the PPS ismaintained at a pH ranging between about 6.4 and about 7.6.
 25. A methodof storing platelets, wherein isolated platelets are obtained from oneor more donors, the method comprises: a. contacting the isolatedplatelets with a PPS that comprises: i. an amount of one or moreβ-galactosidase inhibitors and an amount of one or more sialidaseinhibitors, and optionally an amount of one or more glycan-modifyingagents, or a combination thereof; and ii. one or more of PPS componentsthat includes a salt, a citrate source, a carbon source, or anycombination thereof wherein the one or more PPS components comprises: asodium source in an amount ranging between about 100 mM and about 300mM; a chloride source in an amount ranging between about 40 mM and about110 mM; a citrate source in an amount ranging between about 2 mM andabout 20 mM; an acetate source in an amount ranging between about 10 mMand about 50 mM; a phosphate source in an amount ranging between about 5mM and about 50 mM; a potassium source in an amount ranging betweenabout 0.5 mM and about 10 mM; and a magnesium source in an amountranging between about 0.5 mM and about 5.0 mM.
 26. The method of claim25, further comprising at least one of the group consisting of: a. acalcium source in an amount ranging between about 0.1 mM and about 2.5mM; b. a glucose source in an amount ranging between about 0.1 mM andabout 30 mM; and c. both.
 27. The method of claim 25, wherein thechloride source is present in the amount ranging between about 90 mM andabout 110 mM.
 28. The method of claim 25, wherein the sialidaseinhibitor is selected from the group consisting of: fetuin;2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA);ethyl(3R,4R,5S)-5-amino-4-acetamido-3-(pentan-3-yloxy)-cyclohex-1-ene-1-carboxylate);(2R,3R,4S)-4-guanidino-3-(prop-1-en-2-ylamino)-2-((1R,2R)-1,2,3-trihydroxypropyl)-3,4-dihydro-2H-pyran-6-carboxylicacid;(4S,5R,6R)-5-acetamido-4-carbamimidamido-6-[(1R,2R)-3-hydroxy-2-methoxypropyl]-5,6-dihydro-4H-pyran-2-carboxylicacid;(1S,2S,3S,4R)-3-[(1S)-1-acetamido-2-ethyl-butyl]-4-(diaminomethylideneamino)-2-hydroxy-cyclopentane-1-carboxylicacid; a combination thereof; and a pharmaceutically acceptable saltthereof.
 29. The method of claim 28, wherein the sialidase inhibitor isthe sodium salt of 2,3-dehydro-2-deoxy-N-acetylneuraminic acid.
 30. Themethod of claim 25, wherein the one or more β-galactosidase inhibitorsare selected from the group consisting of: 1-deoxygalactonojirimycin(DGJ); N-(n-butyl)deoxygalactonojirimycin;N-(n-nonyl)deoxygalactonojirimycin; 5-deoxy-L-arabinose; galactostatinbisulfate; 3′,4′,7-trihydroxyisoflavone; D-ribonolactone;N-octyl-4-epi-β-valienamine; phenylethyl β-D-thiogalactopyranoside;difluorotetrahydropyridothiazinone; 4-aminobenzyl1-thio-β-D-galactopryranoside; a combination thereof; and apharmaceutically acceptable salt thereof.
 31. The method of claim 25,wherein the isolated platelets are stored for a period of about 1 toabout 21 days.
 32. The method of claim 25, wherein the isolatedplatelets are stored a temperature of between about 2° C. and about 25°C.
 33. The method of claim 25, further comprising cooling the plateletcomposition to a temperature below room temperature; storing theplatelet composition for a period of time; and then rewarming theplatelet composition back to room temperature.
 34. The method of claim25, further including treating the population of platelets with theβ-galactosidase inhibitor and the sialidase inhibitor, within a timeperiod, wherein the time period is in a range between about 1 minute toabout 8 hours.